Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model

Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model Nikolai Klymiuk, Petra Runa Vochozkova Runa Vochozkova, Michaela Vaskovicova, and 24 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7194258/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Complex diseases such as progressive cardiomyopathies are often insufficiently reflected in small animal and in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation (cGM) has not yet been applied to pig in an effective manner, mainly due to biological and logistic limitations. Here, we describe the generation of a novel humanized model for PLN-mediated cardiomyopathy and compare different methods of activating a pathogenic R14del mutation by Cre-mediated recombination. Both, the Cre- treatment of pig primary cells before somatic cell nuclear transfer as well as the micro-injection of Cre-encoding mRNA into zygotes, were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding AAV into piglets was sufficient in cGM, albeit at to a varying extent. Together, we here describe a highly effective process to establish complex cGM traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventations during reproduction or post-natally. Biological sciences/Biotechnology/Animal biotechnology/Genetic engineering Health sciences/Medical research/Translational research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction An increasing number of cardiomyopathies, of either dilated or hypertrophic or arrhythmogenic phenotype, has been demonstrated to be caused by (likely) pathogenic genetic variants. Penetrance, severity and onset of the phenotype often vary in a patient population and patho-physiological mechanisms involve secondary effects that are seemingly unrelated to the immediate genetic defect. Studying diseases such as arrhythmogenic cardiomyopathies in animal models also involve physiological and pathophysiological mechanisms that differ between model species and humans. Although frequently used for ischemic and hypertrophic cardiomyopathy studies, especially the staggering heart rate and the limited regulatory capacity of frequency make it harder to directly translate results from the mouse species to human studies. In accordance, the mouse heart is rarely tweaked into ventricular or atrial fibrillation due to its high resting frequency, although high doses of caffeine and catecholamines may induce ventricular tachycardias. In contrast, pig hearts offer an appealing alternative. Obviously, depending on age and size, pig hearts can grow to similar or even bigger sizes as human hearts, and share proportion, perfusion and valve mechanics. Though being more vulnerable to arrhythmias, pig hearts are studied well using clinical grade electrophysiologic (EP) catheterization and high-resolution mapping (Moretti et al., 2020). They share the occurrence of ischemia-reperfusion arrhythmias, for which they are even more vulnerable and are susceptible for ventricular tachycardias inflicted by exogenous beating cell transplants (Wulkan et al., 2024). Finally, they succumb to a sudden cardiac death phenotype, when (likely) pathogenic genetic variants are present inflicting areas of low or no-amplitude action potentials in EP maps. Of note, successful treatments preventing structural and intracellular deterioration during the unfolding of a genetic cardiomyopathy also result in electrophysiologic stabilization (Bähr et al., 2023). Increasing value of the pig species even further, the toolbox for genetic modification of the species has expanded dramatically (Boettcher et al., 2022; Holm et al., 2016; Howland et al., 2020; Watson et al., 2016), primarily due to the availability of highly potent CRISPR/Cas-based genome modification tools (Klymiuk et al., 2016; Tanihara et al., 2021; Wu et al., 2018). This allowed for fast engineering, regulation of transgene expression (Jin et al., 2014; Klymiuk et al., 2012a) as well as for establishing complex genetic traits such as the targeting of multiple sites (Anand et al., 2023; Griffith et al., 2025) or the (partial) humanization of disease-relevant genes (Grotz et al., 2022). However, evident and insurmountable biological traits, such as longer reproduction cycles, larger size, maintenance costs, etc, hinder implementation of genetically modified (GM) pigs to a scale as it has been established in mice. This limitation specifically includes conditional GM (cGM) models that allow for switching on genetic function in certain cell types, in defined developmental lineages or at desired time points. Aiming at excision, insertion, inversion or exchange of defined genomic sites, cGM generally involve recombinase enzymes that bind and tie defined recognition sites after their stable integration into a genomic site of interest (Oumard et al., 2006; Turan et al., 2013). Very often, cGM comprise Cre enzymes recognizing lox-segments of 34bp (Kuhn et al., 1995), with alternative use of FLTP-FRT, cphi31-attR or others. In mice, recombinases and their recognition sites are mostly applied as independent GM at distinct genomic localizations, allowing for maximal flexibility and modular combination of cGM systems or multiple modification sites, such as genome-wide gene trapping (Schebelle et al., 2010) or multi-cassette accumulation (Kameyama et al., 2010). Several pig lines involving lox-sites for reporter gene induction (Li et al., 2014a; Li et al., 2014b; Wang et al., 2017) or for oncogene activation (Li et al., 2015; Schook et al., 2015) are available. Although GM pigs expressing Cre-recombinase under PTF1A (Kalla et al., 2021), AQP2 (Luo et al., 2014), VASA (Song et al., 2016), MX1 (Chen et al., 2010) or TYR (Oh et al., 2025) promoters have been presented as well, it is common sense that a concept as has been established in mice, providing systematic supply of a vast variety of Cre-lines at commercial or academic exchange basis cannot be expected for other species. Rather, cGM in pig must rely on alternative strategies. Here, we present a pig model for genetically inherited arrhythmogenic cardiomyopathy, caused by mutated phospholamban (PLN) by the frequent PLN c.40_42delAGA; p.(R14del) pathogenic variant (Nagel et al., 2012), for which we combined deep design approaches with advanced GM strategies and Cre-mediated conditional activation of the pathogenic variant. Results Mining for PLN translational prospect To explore the suitability of Sus scrofa / pig as a model species to investigate PLN dysfunction, we explored the degree of conservation of PLN and its main interacting partners, the SERCA1 (ATP2A1) and SERCA2 (ATP2A2) Ca 2+ pumps. Among representative mammalian species, SERCA proteins show almost complete identity in the sites that have been identified to interfere with PLN ( Fig. S1 ). The short PLN peptide shows strict conservation of the membrane-spanning domain II and loop domain as well as in the regulatory phosphorylation site Ser16 (S16) and Thr17 (T17) (Fig. 1A) . Variations in the regulatory and inhibitory regions of PLN such as p.3D/E, p.7C/H (both domain Ia) and p.27K/N (domain Ib) reflect species-specific components to SERCA-PLN interaction. The consequences of variation for PLN-SERCA interactions have not been examined so far. At transcriptomic level, short- and long-read sequencing in 7 PLN-R14del carriers revealed strict correlation of the PLN c.40_42del variant (rs397516784) with the presumably benign rs12198461 and the rs1051429 single nucleotide variant (SNV) in the 3´-UTR of PLN (Fig. 1B) . This linkage is in line with the hypothesis that a single founder event figures as the origin for the North-Western European PLN-R14del patients (van der Zwaag et al., 2013). Multi-species alignments of the PLN genomic locus showed a relatively low degree of conservation among mammals, compared to previously assessed genes (Grotz et al., 2022). Substantial homology was restricted to the 2 potential enhancer elements upstream of exon 1, the immediate surroundings of exon 1 and the coding region of PLN in exon 2 (Fig.1C) . Notably, the large 3´-UTR shows substantial divergence between mammals besides a few conserved elements. While the immediate poly-adenylation sites are highly conserved. The particular appearance of repetitive elements within the 3´-UTR in several species suggested lower degree of conservation. In human, all SNV allocated to the PLN 3´-UTR were classified as of uncertain significance or likely benign. Thus, we conclude that pig SERCA proteins can be fully regulated by human PLN. However, we presume that human PLN may have distinct regulatory properties than porcine PLN. Furthermore, post-transcriptional regulation might be distinct between pig and human PLN transcripts. PLN modification strategy To mimic the action of human wild-type (WT) and defective PLN in a pig model, we proposed that the GM design must integrate (i) the causative mutation itself, (ii) the entire human PLN protein constituents, and (iii) the main post-transcriptional regulatory properties of the extended 3´-UTR. The pathological course, establishing disease from a single allele, suggests (iv) a fallback strategy for the case that the dominant negative disease expression interferes with reproduction capacities. The post-translational switching between mono- and pentamer, presumably involved in disease shaping, raises questions about the interaction between human and pig PLN. To integrate all these considerations, we aimed to create a PLN-R14del pig model that combines intact and mutated human PLN protein and a human-like post-transcriptional regulation with a potential fallback strategy that allows for targeted activation of the R14del SNV. We employed a GM strategy that initially established a double-cassette construct, comprising a removable exon that produces an intact human PLN protein and a downstream exon encoding a human R14del protein upon removal of the human PLN-WT cassette (Fig. 2A) . Integration of the complete GM into the PLN pig locus was achieved by homologous recombination (HR) while the activation of the PLN-R14del was done by Cre-lox mediated excision of the upstream components. The Cre-lox strategy allowed for the expression of huPLN-WT from the complete construct and the expression of huPLN-R14del from the Cre-modified construct. Combinatorial breeding of the huPLN variants (Fig. 2B) may be used to constitute the patient-relevant huPLN WT/R14del genetic constellation or a homozygous huPLN R14del/R14del variant, presumably leading to an accelerated phenotype (Eijgenraam et al., 2020). Building on previous work, we followed a GM protocol that combined a modified bacterial artificial chromosome (BAC) (Klymiuk et al., 2013; Klymiuk et al., 2012b), carrying the desired GM and CRISPR/Cas9-mediated double-strand breaks (DSB) (Vochozkova et al., 2019) to stimulate HR of the modified BAC with the target region. Modified BACs provide the capacity for large GM such as the complete PLN-WT – neo – PLN-R14del modification and extended regions of homology to the targeting region, avoiding the necessity for isogenic homologous arms. To modify the BAC CH242-318M5, covering the pig PLN locus, we combined gene synthesis, sequential plasmid cloning and bacterial recombineering (Fig. 3A) . Modification of the BAC was confirmed by end-point PCR spanning homologous arms (Fig. 3B) and integrity of the modified BAC was verified by restriction enzyme finger printing (Auch et al., 2022) (Fig. 3C) . To induce HR-stimulating DSB, 3 gRNAs were designed and tested, revealing substantially distinct NHEJ mutation rates (PLN-BACg1: 11-12%, PLN-BACg2: 0% and PLN-BACg3: 40-41%) (Fig. 3.D) . Humanizing the pig PLN locus According to established protocols (Richter 2012) (Fig. 4.A) , primary cell lines of 2 distinct WT pigs were nucleofected with plasmids expressing Cas9, gRNAs and a linearized modification BAC. After clonal selection and passaging, 161 single cell clones (SCC) were cryo-conserved and screened for homologous recombination in a sequential protocol (Fig. 4B) . qPCR-based loss-of-wildtype allele (LoWA) detected 100-6000 genomic copies / µl in the DNA preparations of SCC and rejected 138 clones as potentially modified at the 5´-region of the modification (Fig. 4.C) . One of the 23 candidate SCC did not retain a pig PLN allele, suggesting bi-allelic modification of the target locus. The other candidates were further screened by sequencing a PCR product spanning the CRISPR-gRNA binding site. By assessing 2 naturally occurring SNV in the amplicon as well as the appearance of NHEJ-mediated mutations (Fig. 4D) , 13 SCC were confirmed to retain a single pig PLN allele. Sanger sequencing of the remaining PLN allele in these SCC confirmed that the majority acquired a gap that exactly referred to the cutting sites of the gRNA used to stimulate HR (Fig. 4E) . Alternative NHEJ pattern was observed occasionally. Final validation for abundance of the huPLN-WT, huPLN-R14del and neo-coding regions designated a total of 8 candidate clones for SCNT. For SCNT experiments, SCC were recovered and prepared for SCNT, including the nucleofection with Cre-encoding plasmid or lipofection with Cre-mRNA to activate the huPLN-R14del cassette. 4 SCNT were conducted, using pooled SCC preparations, according to (Kurome et al., 2015). 2 litters delivered 3 viable and 2 stillborn piglets (Fig. 5A) . Genotyping by endpoint PCR (Fig. 5B, C) revealed 3 of them to carry the complete huPLN-WT modification and 2 retaining the huPLN-R14del cassette, indicating Cre-mediated excision after nucleofection with Cre-encoding plasmid. Sanger sequencing of PCR products spanning the modification segment confirmed the abundance and identity of huPLN-WT and huPLN-R14del genetic elements and the correct transition between them (Fig. S3) . Sequencing of the poPLN-WT allele allowed the correlation of founder animals 11809 as well as the majority of re-cloned animals to SCC_111, while the origin of the others remained unclear due to the consistent NHEJ pattern in the other SCC used in SCNT (Fig. 5D, E) . To further explore the genomic constellation of GM elements, animals #11810, #11926 and #12435, a viable clone of #11809, were raised and propagated by a breeding scheme that combines outbreeding for increased fertility and fitness with inbreeding to achieve a huPLN WT/R14del genetic constellation at the PLN locus (Fig. 5F, G) . Quantitative exploration for the poPLN-WT, huPLN-WT, huPLN-R14del (Fig. S4) genetic elements confirmed genotyping by endpoint PCR (Fig. 5H, I) , with genotype proportions according to the Mendelian rules of inheritance. Quantification of the neo selection cassette in F1 animals (Fig. 5J) revealed that the neo element segregated from the PLN-locus in the #11809 lineage and purified PLN-R14del individuals lacking the neo element were selected for further breeding. In line #11926, multiple copies of neo fragments were inherited with the PLN-locus, suggesting an inappropriate genetic modification of the target locus and clearing of this line from further breeding attempts. Activating of huPLN-R14del by mRNA micro-injection In parallel to the cleaning of the PLN-R14del line #11809 from the neo fragment by Mendelian segregation, we aimed at an alternative activation protocol of huPLN-R14del in the pure PLN-WT line #11810, using the Cre-mediated activation in GM zygotes. Sperm of a huPLN-WT F1 boar was used to mate synchronized WT sows of the Libechov minipig breed. Next day, sows were slaughtered, and presumptive zygotes were flushed from oviduct, cultivated in improved defined medium and microinjected with highly pure mRNA carrying a codon-optimized variant of Cre-recombinase, according to published data (Whitworth et al., 2018). To test recombination efficiency, microinjected zygotes were cultured in vitro ( Fig. 6A ) for 4-5 days. The developmental potential of embryos was not compromised by micro-injection as 58.33% of embryos reached morula or blastocyst stage. After termination, 24 embryos at varying stages were used for nested end-point PCR. While the pattern of two PCR products aiming at B2M reference site and pig PLN locus was not fully conclusive, PCR sensitive for the complete modification encoding huPLN-WT or modified cassette encoding huPLN-R14del indicated partial efficacy of Cre-mediated cassette excision after micronjection (Fig. 6B) . Next, micro-injected zygotes were cultivated for one day and then approx. 30 embryos at the 2-4 cell stage were transferred to synchronized gilts. Out of 3 embryo transfers, 1 pregnancy was established, delivering 5 viable offspring (Fig. 6C, D) . Genotyping revealed 1 piglet to be poPLN WT/WT , while 2 piglets carried the non-modified huPLN-WT cassette and 2 others were successfully modified to express the huPLN-R14del cassette. To test mosaicism in MI-derived piglets, Sanger sequencing of PCR products spanning across the first loxP site revealed that piglets MP3 and MP4 had a pattern that unambiguously correlated to the huPLN-R14del genotype, without any sign of remaining huPLN-WT (Fig. 6E) . Likewise, qPCR-based copy number determination on gDNA from skin and blood of the MI-derived piglets revealed a clear huPLN-WT pattern in MP1 and MP5 whereas MP3 and MP4 were huPLN-R14del without any indication of a remaining huPLN-WT pattern (Fig. 6F) . Post-natal activation of huPLN-R14del genotype In a third attempt, we sought to activate the huPLN-R14del cassette in a viable animal. For this, F1 piglets of heterozygous huPLN-WT/poPLNWT or homozygous huPLN-WT/huPLN-WT genotypes injected intravenously at an age of 4-8 weeks with 1.0x10 15 -1.0x10 16 vp of Cre-encoding AAV2/9, with or without PAMAM coating or improved cardiomyocyte transduction. Heart tissue was systematically sampled for the left (LV) and right ventricles (RV) (Fig. 7A) and examined for induction of the huPLN-R14del cassette and virus abundance. One pair of heterozygous animals was kept for 21 weeks injection of AAV without coating. At genomic levels, the Cre efficacy ranged between 0.6-3.8% in representative LV and 0.4-2.1% in representative RV samples, compared to the levels in huPLN-R14del/poPLN-WT animals, whereas the abundance of huPLN-R14del transcripts reached 9.7-27.9% for LV and 0.9-13.4% for RV (Fig. 7B, C) . In an attempt with G2-cys PAMAM coated AAV, another pair of animals indicated increased activation of huPLN-R14del cassettes, correlating with evident amounts of virus genomes in heart tissue samples 3 weeks after administration (Fig. 7D-F) . Coating with G2-myoP8 PAMAM resulted in varying efficacy in homozygous huPLN-WT/huPLN-WT pigs. In one animal, the huPLN-R14del variant made up 16-37% of PLN transcripts, while in another animal the value only reached 2%, whereas the amount of virus genomes was comparable in both animals. Transcriptional constellations at modified PLN loci To validate correct expression of the respective genotypes, transcription of poPLN-WT, huPLN-WT and huPLN-R14del was determined by Sanger sequencing of RT-PCR amplicons spanning poPLN exon 1 and the respective downstream exons. In poPLN WT/WT animals, the exon boundaries and coding sequence (Fig. 8A) were in line with GenBank entry, confirming conserved splice donor and splice acceptor sites in multi-species alignment (Fig. 1C) . In huPLN-WT constellation, poPLN-WT exon 1 was predominantly spliced to huPLN-WT exon 2. At low frequency (<5%) alternative splicing occurred to the downstream huPLN-R14del exon (Fig. 8B) . In huPLN-R14del constellation, the splicing from poPLN-WT exon 1 to huPLN-R14del exon 2 was effective, confirming that splice acceptor sites acted correctly when shuttled between species (Fig. 8C) . Discussion Here, we combine CRISPR-based homologous recombination with Cre-mediated recombinase-mediated cassette excision (RMCE) to establish a complex humanized knock-in pig model for PLN-mediated heart disease. While some of the GM components have been presented by us and others in independent studies earlier, true cGM approaches have not yet been presented before in a systematic manner. In pigs, various GM methods have been tested (Hamze et al., 2025). In the pre-CRISPR era, site-directed modifications were only achieved for a few designated loci, such as GGTA (reviewed in (Klymiuk et al., 2010)), CFTR (Klymiuk et al., 2012b; Rogers et al., 2008) or DMD (Klymiuk et al., 2013). Nowadays they are often established by direct injection of CRISPR/Cas components into zygotes (Fu et al., 2024; Geisert et al., 2023; Maynard et al., 2021; Whitworth et al., 2017), either aiming at the error-prone NHEJ pathway or homology-directed repair (HDR), using single strand deoxy-nucleotides (ssODN) to introduce a defined mutation (Klymiuk et al., 2016). Larger site-directed modifications, however, still appear challenging and are commonly established and verified in primary cells (Vochozkova et al., 2019), before they are used as nucleus donor in SCNT (Kurome et al., 2015). Most large GM attempts build on targeting vectors that flank the desired GM with homologous arms of several 100bp up to few kb lengths, whereas we have gained vast experience in manipulating BACs for becoming targeting vectors. Initially created to clone segments of up to 200kb eukaryotic DNA into plasmid vectors during genome sequencing projects, BACs represent a resource that can be modified extensively by bacterial recombineering in dedicated E.coli strains (Auch et al., 2022), giving rise to vector with huge genomic regions that serve as homologous arms during recombination with the target region in pig primary cells (Beshr et al., 2017; Klymiuk et al., 2013; Klymiuk et al., 2012b). While chromatin accessibility is commonly thought as critical for GM efficacy (Chari et al., 2015; Jensen et al., 2017), combinations of BAC vectors with CRISPR/Cas-mediated DSB performed well on target sites that were considered silent in pig primary cells, such as USH1C (Grotz et al., 2022) or PLN, as we demonstrated here. Contrary to the widely used technologies for constitutive GM in defined genomic sites, methods that allow for cGM remain sparse in pigs, not to speak of dissemination as it has been seen in mouse. Initially, Cre-mediated cassette excision of genetic modifications (Li et al., 2014a; Moon et al., 2012) demonstrated proof-of-concept in pig cells in vitro, but only few reports demonstrated RMCE in vivo (Moretti et al., 2020; Schook et al., 2015). The generation of numerous pig strains expressing Cre under certain conditions (Chen et al., 2010; Kalla et al., 2021; Luo et al., 2014; Oh et al., 2025; Song et al., 2016) suggests that the reason behind this is not merely scientific. Indeed, distribution or share of existing GM pig lines between research groups has been rather confined so far, and we see little perspective for an exchange system that facilitates crossbreeding GM strains with Cre-lines upon request in a foreseeable future. With advancing biomedical models in pig, however, cGM approaches will become more relevant, as we exemplify here for a model for PLN-R14del arrhythmogenic cardiomyopathy. The chosen strategy involved several considerations: While aberrant phosphorylation of the mutated protein alone enhance SERCA inhibition by direct interaction (Vafiadaki et al., 2022; Vostrikov et al., 2015), the assembly of PLN into pentamers is highly relevant for its regulatory properties (Funk et al., 2023; Glaves et al., 2019; Smeazzetto et al., 2017; Traaseth et al., 2007; Wittmann et al., 2015)), suggesting direct interaction of WT and mutated PLN proteins. For the difference in pig and human PLN, the option to combine human PLN-R14del with human PLN-WT was therefore seen as precondition. Further, the dominant negative pathophysiology raised concerns about detrimental effects on reproduction, in case of early disease onset, suggesting a cGM approach as fallback strategy. Finally, SCNT of primary cells remains a bottleneck technology in biomedical engineering, as it critically depends on restricted and seasonal cloning capacities, on the very source of primary cells or the developmental capacities of validated single cell clones (Kurome et al., 2013; Richter et al., 2012). The introduction of a complex but single cGM into one allele of the target locus in primary cells by gene editing and its subsequent modulation meticulously balanced risk with effort in our GM pipeline. The combination of gene synthesis and plasmid cloning allowed for efficient construction of a 9kb modifications construct and its efficient integration into a BAC covering the pig PLN gene (Fig. 3). Human PLN-WT was expressed from the constitutive stage of the GM while Cre-mediated RMCE effectively induced the R14del genotype (Fig. 7). Once established, human WT and R14del variants of the modified PLN gene were combined by crossbreeding to precisely re-constitute the dynamics between human PLN-WT and R14del in mono- and oligomers (Fig. 2B). Further, the comparison of distinct Cre application strategies to activate the PLN-R14del cassette illustrated the capacities to establish cGM in pig without the necessity to create a complementary GM line expressing Cre as transgene. The treatment of verified SCC with Cre-encoding mRNA, via lipofection, or plasmid nucleofection prior to SCNT elegantly established both, huPLN-WT and huPLN-R14del genetic variants within a single cloning experiment (Fig. 5C). Success rates of the SCNT procedure and the number of viable founder piglets are remarkable, recapitulating similar efficacy with the same (Grotz et al., 2022) and other primary cell lines (Stirm et al., 2023). The thriving re-cloning capacities of primary cells from a deceased PLN-R14del founder individual (#11809) further confirmed the used primary cell line as highly potent for GM and SCNT. Indeed, extended genotyping (Fig. 5H, S2) demonstrated that several cell clones out of the verified SCC pool developed to founder animals. Together, SCNT is a powerful tool to establish complex GM in pig and combination of gene editing and Cre-recombinase facilitates the generation of constitutive and activated variants of cGM in one go, if all components of primary cell manipulation, in vitro reproductive technologies and embryo transfer were properly executed. Direct micro-injection of Cre-mRNA into the cytoplasm of cultivated embryos that were produced with sperm of a boar containing the entire cGM cassette (Fig. 6), represented a valuable alternative to SCNT of Cre-treated primary cells. In contrast to SCNT, which facilitates the generation of genetically identical clones from a thoroughly characterized primary cell population, IVF-derived pigs resemble littermates because of germ lines meiosis. Thus, parental alleles are inherited in a ratio of 1:1 and only 50% offspring carry a desired genetic trait, if this appeared in a mono-allelic constellation in one of the parents. From a pragmatic point of view, this genetic drawback is often outcompeted by the beneficial reproductive features as IVF appears technically more robust and yields higher developmental potential than SCNT embryos (Isom et al., 2012; Yamanaka et al., 2009; Zhai et al., 2022), correlating to larger litter sizes and, importantly, better vitality of the piglets even after embryo micromanipulation (Chen et al., 2022). In our approach, we further stimulated the developmental potential by flushing embryos from the oviducts of inseminated sows, avoiding the lower developmental competence associated to the use of in vitro produced embryos (Bauer et al., 2010; Cambra et al., 2021; Chen et al., 2022). Relevantly, the manipulation of zygotes is often associated with mosaicism, i.e. the activity of the injected compound after the first cleavage of the embryo, resulting in distinct genetic constellations in the cells of an organism. This problem has been seen to a particular extent after injecting CRISPR/Cas components into mouse (Hashimoto et al., 2016), bovine (Wei et al., 2022) or pig zygotes (Navarro-Serna et al., 2021; Whitworth et al., 2017). The completely different mode of action of Cre recombinase and its much faster kinetics, however minimized this risk in mouse zygote injection (Araki et al., 1995; Luckow et al., 2009), which is in line with the unambiguous huPLN-WT and huPLN-R14del genotypes in both blastocysts and piglets derived by micro-injection (Fig. 6E, F). Finally, post-natal activation or the R14del cassette revealed significant cGM efficiencies by intravenous application of a Cre-encoding AAV into young piglets. At first sight, the discrepancies between relatively low Cre-efficacy at genomic and much higher activation at transcript level appear puzzling. Considering, however, that cardiomyocytes were the preferred target of AAV2/9 vectors and likewise also the sole source of PLN transcripts in the heart, transcriptional assessment provides true evidence for the desired cGM activation events. Assuming that there was no regulatory interaction between huPLN-WT and huPLN-R14del alleles, the quantification of PLN transcripts provides additional insight: Any increase in R14del expression indicates a nuclear event and allows for the precise quantification of Cre-mediated events, independent of the nuclear status of cardiomyocytes. While combining Cre-application with reproduction technologies provides a “clean” genotype in the whole organism, the mosaic cGM pattern after post-natal Cre-application, provides the perspective for studying spatial effects, in particular if application can be done at high concentrations during restricted local administration (Lampela et al., 2024). Given the often focal development of cardiomyopathies, this approach may provide fundamentally new insight into disease-forming events and the pathophysiological mechanisms behind their dissemination across larger areas of the heart. Evidently, post-natal application remains the sole option for cGM activation in case of embryonal or fetal fatal genetic disorders. Together, we present a comprehensive design of a cGM pig model for arrhythmogenic cardiomyopathies and document effective ways of cGM activation by applying Cre recombinase at distinct developmental stages. Our findings pave the way for novel future cGM approaches in pig but also in other species for which the complementary transgenic Cre-driving lines are lacking and their development, maintenance and distribution is challenging. Materials and Methods All procedures involving human patients and animals were approved and supervised by the respective local authorities. Detailed Methods are described in a supplementary document. Declarations Author contributions PRV contributed to the design of the model, performed genetic manipulation of pig genome and contributed to the characterization of animals. MV contributed to zygote microinjection. JMC designed and directed and mainly performed characterization of animals and embryos. AB designed and mainly preformed animal intervention and directed breeding program. EMM contributed to animal intervention, characterization of animals and breeding program. MK contributed to SCNT and provided parthenogenic embryos. VZ , TG contributed to SCNT. BK contributed to SCNT and performed embryo transfer. MLC and HCH contributed to animal intervention. TB , VR , IML provided AAV and explored transduction efficacy. PvT , MH explored patient material. SJ and JJ performed embryo transfer. CGCG contributed to animal characterization and breeding management. ZE, JM contributed to breeding management, directed embryo flushing and contributed to the design of the manuscript. PAD , PCG contributed to the design of the model. DD directed and contributed to zygote microinjection. EW contributed to the design of the model and provided SCNT technology. CK contributed to the design of the model and to the writing of the manuscript. NK led overall work, design of the model, characterization and writing of the manuscript. References Anand, R.P., Layer, J.V., Heja, D., Hirose, T., Lassiter, G., Firl, D.J., Paragas, V.B., Akkad, A., Chhangawala, S., Colvin, R.B. , et al. (2023). Design and testing of a humanized porcine donor for xenotransplantation. Nature 622 , 393-401. Araki, K., Araki, M., Miyazaki, J., and Vassalli, P. (1995). Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A 92 , 160-164. Auch, H., Klymiuk, N., and Runa-Vochozkova, P. (2022). Modifying Bacterial Artificial Chromosomes for Extended Genome Modification. Methods Mol Biol 2495 , 67-90. Bähr, A., Hoppmann, P., Bozoglu, T., Stirm, M., Luksch, I., Ziegler, T., Hornaschewitz, N., Shrestha, S., Shashikadze, B., Stöckl, J. , et al. (2023). Gene Therapy for Cardiomyopathy associated with Duchenne Muscular Dystrophy in a Pig Model. bioRxiv, 2023.2010.2002.560452. Bauer, B.K., Isom, S.C., Spate, L.D., Whitworth, K.M., Spollen, W.G., Blake, S.M., Springer, G.K., Murphy, C.N., and Prather, R.S. (2010). Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos. Biol Reprod 83 , 791-798. Beshr, G., Sikandar, A., Jemiller, E.M., Klymiuk, N., Hauck, D., Wagner, S., Wolf, E., Koehnke, J., and Titz, A. (2017). Photorhabdus luminescens lectin A (PllA): A new probe for detecting alpha-galactoside-terminating glycoconjugates. J Biol Chem 292 , 19935-19951. Boettcher, A.N., Schachtschneider, K.M., Schook, L.B., and Tuggle, C.K. (2022). Swine models for translational oncological research: an evolving landscape and regulatory considerations. Mamm Genome 33 , 230-240. Cambra, J.M., Martinez, E.A., Rodriguez-Martinez, H., Gil, M.A., and Cuello, C. (2021). Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium. Animals (Basel) 11 . Chari, R., Mali, P., Moosburner, M., and Church, G.M. (2015). Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods 12 , 823-826. Chen, L., Li, L., Pang, D., Li, Z., Wang, T., Zhang, M., Song, N., Yan, S., Lai, L.X., and Ouyang, H. (2010). Construction of transgenic swine with induced expression of Cre recombinase. Animal 4 , 767-771. Chen, P.R., Uh, K., Redel, B.K., Reese, E.D., Prather, R.S., and Lee, K. (2022). Production of Pigs From Porcine Embryos Generated in vitro. Frontiers in Animal Science Volume 3 - 2022 . Eijgenraam, T.R., Boukens, B.J., Boogerd, C.J., Schouten, E.M., van de Kolk, C.W.A., Stege, N.M., Te Rijdt, W.P., Hoorntje, E.T., van der Zwaag, P.A., van Rooij, E. , et al. (2020). The phospholamban p.(Arg14del) pathogenic variant leads to cardiomyopathy with heart failure and is unreponsive to standard heart failure therapy. Sci Rep 10 , 9819. Fu, B., Ma, H., Huo, X., Zhu, Y., and Liu, D. (2024). CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications. Biomolecules 14 . Funk, F., Kronenbitter, A., Hackert, K., Oebbeke, M., Klebe, G., Barth, M., Koch, D., and Schmitt, J.P. (2023). Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation. Cardiovasc Res 119 , 1568-1582. Geisert, R.D., Johns, D.N., Pfeiffer, C.A., Sullivan, R.M., Lucas, C.G., Simintiras, C.A., Redel, B.K., Wells, K.D., Spencer, T.E., and Prather, R.S. (2023). Gene editing provides a tool to investigate genes involved in reproduction of pigs. Mol Reprod Dev 90 , 459-468. Glaves, J.P., Primeau, J.O., Espinoza-Fonseca, L.M., Lemieux, M.J., and Young, H.S. (2019). The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA. Biophys J 116 , 633-647. Griffith, B.P., Grazioli, A., Singh, A.K., Tully, A., Galindo, J., Saharia, K.K., Shah, A., Strauss, E.R., Odonkor, P.N., Williams, B. , et al. (2025). Transplantation of a genetically modified porcine heart into a live human. Nat Med 31 , 589-598. Grotz, S., Schafer, J., Wunderlich, K.A., Ellederova, Z., Auch, H., Bahr, A., Runa-Vochozkova, P., Fadl, J., Arnold, V., Ardan, T. , et al. (2022). Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes. EMBO Mol Med 14 , e14817. Hamze, J.G., Cambra, J.M., Navarro-Serna, S., and Martinez-Serrano, C.A. (2025). Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives. Genomics 117 , 111014. Hashimoto, M., Yamashita, Y., and Takemoto, T. (2016). Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol 418 , 1-9. Holm, I.E., Alstrup, A.K., and Luo, Y. (2016). Genetically modified pig models for neurodegenerative disorders. J Pathol 238 , 267-287. Howland, D., Ellederova, Z., Aronin, N., Fernau, D., Gallagher, J., Taylor, A., Hennebold, J., Weiss, A.R., Gray-Edwards, H., and McBride, J. (2020). Large Animal Models of Huntington's Disease: What We Have Learned and Where We Need to Go Next. J Huntingtons Dis 9 , 201-216. Isom, S.C., Li, R.F., Whitworth, K.M., and Prather, R.S. (2012). Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis. Mol Reprod Dev 79 , 197-207. Jensen, K.T., Floe, L., Petersen, T.S., Huang, J., Xu, F., Bolund, L., Luo, Y., and Lin, L. (2017). Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency. FEBS Lett 591 , 1892-1901. Jin, Y.X., Jeon, Y., Lee, S.H., Kwon, M.S., Kim, T., Cui, X.S., Hyun, S.H., and Kim, N.H. (2014). Production of pigs expressing a transgene under the control of a tetracycline-inducible system. PLoS One 9 , e86146. Kalla, D., Flisikowski, K., Yang, K., Sanguesa, L.B., Kurome, M., Kessler, B., Zakhartchenko, V., Wolf, E., Lickert, H., Saur, D. , et al. (2021). The Missing Link: Cre Pigs for Cancer Research. Front Oncol 11 , 755746. Kameyama, Y., Kawabe, Y., Ito, A., and Kamihira, M. (2010). An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange. Biotechnol Bioeng 105 , 1106-1114. Klymiuk, N., Aigner, B., Brem, G., and Wolf, E. (2010). Genetic modification of pigs as organ donors for xenotransplantation. Mol Reprod Dev 77 , 209-221. Klymiuk, N., Blutke, A., Graf, A., Krause, S., Burkhardt, K., Wuensch, A., Krebs, S., Kessler, B., Zakhartchenko, V., Kurome, M. , et al. (2013). Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Hum Mol Genet 22 , 4368-4382. Klymiuk, N., Bocker, W., Schonitzer, V., Bahr, A., Radic, T., Frohlich, T., Wunsch, A., Kessler, B., Kurome, M., Schilling, E. , et al. (2012a). First inducible transgene expression in porcine large animal models. FASEB J 26 , 1086-1099. Klymiuk, N., Mundhenk, L., Kraehe, K., Wuensch, A., Plog, S., Emrich, D., Langenmayer, M.C., Stehr, M., Holzinger, A., Kroner, C. , et al. (2012b). Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl) 90 , 597-608. Klymiuk, N., Seeliger, F., Bohlooly, Y.M., Blutke, A., Rudmann, D.G., and Wolf, E. (2016). Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies. Toxicol Pathol 44 , 346-357. Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995). Inducible gene targeting in mice. Science 269 , 1427-1429. Kurome, M., Geistlinger, L., Kessler, B., Zakhartchenko, V., Klymiuk, N., Wuensch, A., Richter, A., Baehr, A., Kraehe, K., Burkhardt, K. , et al. (2013). Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set. BMC Biotechnol 13 , 43. Kurome, M., Kessler, B., Wuensch, A., Nagashima, H., and Wolf, E. (2015). Nuclear transfer and transgenesis in the pig. Methods Mol Biol 1222 , 37-59. Lampela, J., Pajula, J., Jarvelainen, N., Siimes, S., Laham-Karam, N., Kivela, A., Mushimiyimana, I., Nurro, J., Hartikainen, J., and Yla-Herttuala, S. (2024). Caridac vein retroinjections provide an efficient approach for global left ventricular gene transfer with adenovirus and adeno-associated virus. Sci Rep 14 , 1467. Li, S., Edlinger, M., Saalfrank, A., Flisikowski, K., Tschukes, A., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A. , et al. (2015). Viable pigs with a conditionally-activated oncogenic KRAS mutation. Transgenic Res 24 , 509-517. Li, S., Flisikowska, T., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A., Wolf, E., Flisikowski, K., and Schnieke, A. (2014a). Dual fluorescent reporter pig for Cre recombination: transgene placement at the ROSA26 locus. PLoS One 9 , e102455. Li, X., Yang, Y., Bu, L., Guo, X., Tang, C., Song, J., Fan, N., Zhao, B., Ouyang, Z., Liu, Z. , et al. (2014b). Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res 24 , 501-504. Luckow, B., Hanggli, A., Maier, H., Chilla, S., Loewe, R.P., Dehmel, S., Schlondorff, D., Loetscher, P., Zerwes, H.G., and Muller, M. (2009). Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice. Genesis 47 , 545-558. Luo, W., Li, Z., Huang, Y., Han, Y., Yao, C., Duan, X., Ouyang, H., and Li, L. (2014). Generation of AQP2-Cre transgenic mini-pigs specifically expressing Cre recombinase in kidney collecting duct cells. Transgenic Res 23 , 365-375. Maynard, L.H., Humbert, O., Peterson, C.W., and Kiem, H.P. (2021). Genome editing in large animal models. Mol Ther 29 , 3140-3152. Moon, J., Kim, S., Park, H., Kang, J., Park, S., Koo, O., da Torre, B.R., Saadeldin, I.M., Lee, B., and Jang, G. (2012). Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP. Zygote 20 , 423-425. Moretti, A., Fonteyne, L., Giesert, F., Hoppmann, P., Meier, A.B., Bozoglu, T., Baehr, A., Schneider, C.M., Sinnecker, D., Klett, K. , et al. (2020). Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nat Med 26 , 207-214. Nagel, B., Gruber-Sedlmayr, U., Uhrig, S., Stollberger, C., Klopocki, E., and Finsterer, J. (2012). Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants. BMC Med Genet 13 , 60. Navarro-Serna, S., Hachem, A., Canha-Gouveia, A., Hanbashi, A., Garrappa, G., Lopes, J.S., Paris-Oller, E., Sarrias-Gil, L., Flores-Flores, C., Bassett, A. , et al. (2021). Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination. CRISPR J 4 , 132-146. Oh, D., Hong, N., Eun, K., Lee, J., Cai, L., Kim, M., Choi, H., Jawad, A., Ham, J., Park, M.G. , et al. (2025). Generation of a genetically engineered porcine melanoma model featuring oncogenic control through conditional Cre recombination. Sci Rep 15 , 1616. Oumard, A., Qiao, J., Jostock, T., Li, J., and Bode, J. (2006). Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology. Cytotechnology 50 , 93-108. Richter, A., Kurome, M., Kessler, B., Zakhartchenko, V., Klymiuk, N., Nagashima, H., Wolf, E., and Wuensch, A. (2012). Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol 12 , 84. Rogers, C.S., Stoltz, D.A., Meyerholz, D.K., Ostedgaard, L.S., Rokhlina, T., Taft, P.J., Rogan, M.P., Pezzulo, A.A., Karp, P.H., Itani, O.A. , et al. (2008). Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321 , 1837-1841. Schebelle, L., Wolf, C., Stribl, C., Javaheri, T., Schnutgen, F., Ettinger, A., Ivics, Z., Hansen, J., Ruiz, P., von Melchner, H. , et al. (2010). Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps. Nucleic Acids Res 38 , e106. Schook, L.B., Collares, T.V., Hu, W., Liang, Y., Rodrigues, F.M., Rund, L.A., Schachtschneider, K.M., Seixas, F.K., Singh, K., Wells, K.D. , et al. (2015). A Genetic Porcine Model of Cancer. PLoS One 10 , e0128864. Smeazzetto, S., Armanious, G.P., Moncelli, M.R., Bak, J.J., Lemieux, M.J., Young, H.S., and Tadini-Buoninsegni, F. (2017). Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA. J Biol Chem 292 , 21330-21339. Song, Y., Lai, L., Li, L., Huang, Y., Wang, A., Tang, X., Pang, D., Li, Z., and Ouyang, H. (2016). Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig. FEBS Open Bio 6 , 50-55. Stirm, M., Shashikadze, B., Blutke, A., Kemter, E., Lange, A., Stockl, J.B., Jaudas, F., Laane, L., Kurome, M., Kessler, B. , et al. (2023). Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52. Proc Natl Acad Sci U S A 120 , e2301250120. Tanihara, F., Hirata, M., and Otoi, T. (2021). Current status of the application of gene editing in pigs. J Reprod Dev 67 , 177-187. Traaseth, N.J., Verardi, R., Torgersen, K.D., Karim, C.B., Thomas, D.D., and Veglia, G. (2007). Spectroscopic validation of the pentameric structure of phospholamban. Proc Natl Acad Sci U S A 104 , 14676-14681. Turan, S., Zehe, C., Kuehle, J., Qiao, J., and Bode, J. (2013). Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. Gene 515 , 1-27. Vafiadaki, E., Haghighi, K., Arvanitis, D.A., Kranias, E.G., and Sanoudou, D. (2022). Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca(2+) Handling and Arrhythmogenesis. Int J Mol Sci 23 . van der Zwaag, P.A., van Rijsingen, I.A., de Ruiter, R., Nannenberg, E.A., Groeneweg, J.A., Post, J.G., Hauer, R.N., van Gelder, I.C., van den Berg, M.P., van der Harst, P. , et al. (2013). Recurrent and founder mutations in the Netherlands-Phospholamban p.Arg14del mutation causes arrhythmogenic cardiomyopathy. Neth Heart J 21 , 286-293. Vochozkova, P., Simmet, K., Jemiller, E.M., Wunsch, A., and Klymiuk, N. (2019). Gene Editing in Primary Cells of Cattle and Pig. Methods Mol Biol 1961 , 271-289. Vostrikov, V.V., Soller, K.J., Ha, K.N., Gopinath, T., and Veglia, G. (2015). Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions. Biochim Biophys Acta 1848 , 315-322. Wang, K., Jin, Q., Ruan, D., Yang, Y., Liu, Q., Wu, H., Zhou, Z., Ouyang, Z., Liu, Z., Zhao, Y. , et al. (2017). Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing. Genome Res 27 , 2061-2071. Watson, A.L., Carlson, D.F., Largaespada, D.A., Hackett, P.B., and Fahrenkrug, S.C. (2016). Engineered Swine Models of Cancer. Front Genet 7 , 78. Wei, J., Brophy, B., Cole, S.A., Moormann, J., Boch, J., and Laible, G. (2022). Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos. Front Genet 13 , 925913. Whitworth, K.M., Benne, J.A., Spate, L.D., Murphy, S.L., Samuel, M.S., Murphy, C.N., Richt, J.A., Walters, E., Prather, R.S., and Wells, K.D. (2017). Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs. Transgenic Res 26 , 97-107. Whitworth, K.M., Cecil, R., Benne, J.A., Redel, B.K., Spate, L.D., Samuel, M.S., Prather, R.S., and Wells, K.D. (2018). Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs. Transgenic Res 27 , 167-178. Wittmann, T., Lohse, M.J., and Schmitt, J.P. (2015). Phospholamban pentamers attenuate PKA-dependent phosphorylation of monomers. J Mol Cell Cardiol 80 , 90-97. Wu, H., Liu, Q., Shi, H., Xie, J., Zhang, Q., Ouyang, Z., Li, N., Yang, Y., Liu, Z., Zhao, Y. , et al. (2018). Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci 75 , 3593-3607. Wulkan, F., Romagnuolo, R., Qiang, B., Valdman Sadikov, T., Kim, K.P., Quesnel, E., Jiang, W., Andharia, N., Weyers, J.J., Ghugre, N.R. , et al. (2024). Stem cell-derived cardiomyocytes expressing a dominant negative pacemaker HCN4 channel do not reduce the risk of graft-related arrhythmias. Front Cardiovasc Med 11 , 1374881. Yamanaka, K., Sugimura, S., Wakai, T., Kawahara, M., and Sato, E. (2009). Difference in sensitivity to culture condition between in vitro fertilized and somatic cell nuclear transfer embryos in pigs. J Reprod Dev 55 , 299-304. Zhai, Y., Yu, H., An, X., Zhang, Z., Zhang, M., Zhang, S., Li, Q., and Li, Z. (2022). Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation - a comparative analysis between early porcine embryos and their counterparts in other three mammalian species. BMC Genomics 23 , 772. Additional Declarations Yes there is potential Competing Interest. PCG, PRV, EW, NK are holder of a patent on the PLN pig model Supplementary Files FigS1.tif Fig. S1. PLN-SERCA interactions.Interacting amino acid positions in the SERCA1 (ATP2A1 gene) and SERCA2 (ATP2A2 gene) proteins are highlighted in mammalian species (upper panel) and correlated to the binding region in PLN, as identified in the structural biology studies of Seidel-ref (Seidel 2008), Gorski-ref (Gorski 2015), Glaves-ref (Glaves 2019) and Primeau-ref (Primeau 2018). The respective human amino acid sequence is shown as reference. The positions in other species are dots, if identical, or indicated, if varying. FigS2.tif Fig. S2. Re-nucleofection of SCC. Due to the small amount of available cells in SCC portions, nucleofection was optimized by testing Amaxa/Lonza programs with reduced (5x10 5 ) cells. Evaluation was done in fluorescence microscopy 48 hours after nucleofection (upper panel). Programs CA-137 and EH-100 were further examined by flow cytometry (lower left) to quantify nucleofection and in bright field microscopy (lower right) to determine viability. FigS3.tif Fig. S3. Sequence details of PLN founder animals. Localization and specificity of PCR products on genomic DNA in huPLN-WT/poPLN-WT and huPLN-R14del/poPLN-WT founder animals are indicated (top panel). Representative Sanger electropherograms are shown. Orange: spanning first lox site, indicating the appearance of a 2xG motif in huPLN-R14del (#11809) vs 3xG in huPLN-WT (#11810). Blue: spanning open reading frame, indicating that huPLN-WT #11810 comprise both cassettes (mixed pattern with R14 codon), whereas huPLN-R14del retains only the downstream cassette. Amino acid sequences are indicated in one-letter code. Transition from huPLN-WT to neo cassette (pink) and transition from neo cassette to huPLN-R14del cassette (yellow) only appear in huPLN-WT animal #11810. FigS4.tif Fig. S4. Quantfication of genetic elements during breeding. Animals of representative lines in Fig. 5B were analyzed for the respective genetic elements (A). Copy number ratios are given for poPLN-WT (B), huPLN-WT (C), huPLN-R14del (D) constellations, using NANOG and USH1C as genomic reference loci. MMdraft250715.docx Supplementary Materials and Methods Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7194258","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":520538333,"identity":"5e8e0e4f-4b4c-4342-ab7a-c31212e7eedd","order_by":0,"name":"Nikolai Klymiuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFACNsYDDAwSDBLsDSAeM1FaGCBaeA6TpgWoSSKZSC3mDGwJh3lzLBJnznx/TOoGg7UcQS2WDWwHDvNuk0icLZ3MJp3DkG5MUIvBAfYGsJZ5EC2HExuI1yJ5GKylnggtMIdJMIO1JBB22GG2hINzt0kYz+xJNrbOMUg3JGzL8TbDB2+31cnOOH7w4e2cCmt5graAIoKJB2ECYQ1gwPiDSIWjYBSMglEwQgEAkiM3p+p34EUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3532-1659","institution":"TU Munich","correspondingAuthor":true,"prefix":"","firstName":"Nikolai","middleName":"","lastName":"Klymiuk","suffix":""},{"id":520538334,"identity":"fffe34d0-9e76-476e-9f2a-9f2d19709124","order_by":1,"name":"Petra Runa Vochozkova Runa Vochozkova","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Petra","middleName":"Runa Vochozkova Runa","lastName":"Vochozkova","suffix":""},{"id":520538335,"identity":"f254e01e-5f75-41e2-b5a9-5851c4b47db0","order_by":2,"name":"Michaela Vaskovicova","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"Michaela","middleName":"","lastName":"Vaskovicova","suffix":""},{"id":520538336,"identity":"7d821e98-ed4f-4bf6-97a2-67768864a7ab","order_by":3,"name":"Josep Miquel Cambra Bort","email":"","orcid":"https://orcid.org/0000-0003-2010-5849","institution":"Technical University of Munich","correspondingAuthor":false,"prefix":"","firstName":"Josep","middleName":"Miquel Cambra","lastName":"Bort","suffix":""},{"id":520538337,"identity":"f21332ab-393a-4170-9a50-e4a4419625d4","order_by":4,"name":"Andrea Bähr","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Bähr","suffix":""},{"id":520538338,"identity":"7ba08c3f-254a-4dec-8282-f6d1b7b4c98f","order_by":5,"name":"Eva-Maria Männer","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Eva-Maria","middleName":"","lastName":"Männer","suffix":""},{"id":520538339,"identity":"7c13e886-a3b3-4c7e-a8a4-63bde7011390","order_by":6,"name":"Mayuko Kurome","email":"","orcid":"","institution":"LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Mayuko","middleName":"","lastName":"Kurome","suffix":""},{"id":520538340,"identity":"72cff0a8-128c-45cf-95f8-5cc7dc8783f7","order_by":7,"name":"Valeri Zakhartchenko","email":"","orcid":"","institution":"LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Valeri","middleName":"","lastName":"Zakhartchenko","suffix":""},{"id":520538341,"identity":"a73a8056-0da3-4a19-85ef-5ac9c7c03929","order_by":8,"name":"Barbara Kessler","email":"","orcid":"","institution":"LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Kessler","suffix":""},{"id":520538342,"identity":"2aab9ba4-a5c6-4811-b88e-698bb7b2673d","order_by":9,"name":"Tuna Güngör","email":"","orcid":"","institution":"LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Tuna","middleName":"","lastName":"Güngör","suffix":""},{"id":520538343,"identity":"99e1806c-d53c-4b9f-b541-11dd2ac85684","order_by":10,"name":"Mara Corsten","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Mara","middleName":"","lastName":"Corsten","suffix":""},{"id":520538344,"identity":"63c6e8be-1fee-4700-8826-24ea44046ee6","order_by":11,"name":"Heinke Heymer","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Heinke","middleName":"","lastName":"Heymer","suffix":""},{"id":520538345,"identity":"c15f33d6-6512-4f51-ba9e-56736e670d88","order_by":12,"name":"Tarik Bozoglu","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Tarik","middleName":"","lastName":"Bozoglu","suffix":""},{"id":520538346,"identity":"9b2fa04f-0773-410e-be49-ce1e5d48e5f5","order_by":13,"name":"Vijayanand Rajendran","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Vijayanand","middleName":"","lastName":"Rajendran","suffix":""},{"id":520538347,"identity":"a9fada7e-116b-48cb-a8b6-c8e9492b6b78","order_by":14,"name":"Ina Luksch","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Ina","middleName":"","lastName":"Luksch","suffix":""},{"id":520538348,"identity":"1e703882-0fc7-451d-ad1a-1ecff931ece7","order_by":15,"name":"Magdalena Harakalova","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Harakalova","suffix":""},{"id":520538349,"identity":"7546907f-f510-4757-b25a-83174c0f85fd","order_by":16,"name":"J. Peter van Tintelen","email":"","orcid":"https://orcid.org/0000-0003-3854-6749","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Peter van","lastName":"Tintelen","suffix":""},{"id":520538350,"identity":"fe8bbc1f-2a41-425e-bea4-38b486b86fa0","order_by":17,"name":"Stefan Juhas","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Juhas","suffix":""},{"id":520538351,"identity":"1ccb430a-ed9f-41c9-9061-f0fa7c74d3d8","order_by":18,"name":"jana Juhasova","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"jana","middleName":"","lastName":"Juhasova","suffix":""},{"id":520538352,"identity":"c82cad67-915d-466e-a90a-9688694a1b78","order_by":19,"name":"Carolina Cabrera-Gomez","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"","lastName":"Cabrera-Gomez","suffix":""},{"id":520538353,"identity":"ce54cbdd-8b76-438f-b31c-e3311eaaf460","order_by":20,"name":"Daniel Drutovic","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Drutovic","suffix":""},{"id":520538354,"identity":"dc9c858a-b1e9-4e7c-9eab-7c418056f80a","order_by":21,"name":"Zdenka Ellederova","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"Zdenka","middleName":"","lastName":"Ellederova","suffix":""},{"id":520538355,"identity":"22a6a9b7-e03d-4d79-94b6-10d1110f28d8","order_by":22,"name":"Jan Motlik","email":"","orcid":"","institution":"IAPG Libechov","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Motlik","suffix":""},{"id":520538356,"identity":"b5191b53-f532-4a0d-a9ba-2d7a09b797e7","order_by":23,"name":"Pieter Doevendans","email":"","orcid":"","institution":"UMC Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Pieter","middleName":"","lastName":"Doevendans","suffix":""},{"id":520538357,"identity":"171b5028-0f1c-41dd-b711-a69943d3ffe8","order_by":24,"name":"Eckhard Wolf","email":"","orcid":"https://orcid.org/0000-0002-0430-9510","institution":"Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-University Munich","correspondingAuthor":false,"prefix":"","firstName":"Eckhard","middleName":"","lastName":"Wolf","suffix":""},{"id":520538358,"identity":"4f5f145b-3ae1-4eac-a27e-1545dc7cd121","order_by":25,"name":"Pieter Glijnis","email":"","orcid":"","institution":"PLN Foundation","correspondingAuthor":false,"prefix":"","firstName":"Pieter","middleName":"","lastName":"Glijnis","suffix":""},{"id":520538359,"identity":"ef8fa7d8-e62e-4a8b-9510-11b056a8ee9b","order_by":26,"name":"Christian Kupatt","email":"","orcid":"","institution":"TU Munich","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Kupatt","suffix":""}],"badges":[],"createdAt":"2025-07-23 08:56:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7194258/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7194258/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92252192,"identity":"586ef285-504a-4262-aa3d-ce1fd7a0de0e","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2910085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranslational assessment of PLN.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Alignment of mammalian PLN, with human as reference. Only differing positions are shown for other species. Domains are indicated according to Gustavsson-ref (Gustavsson 2013 doi:10.1073/pnas.1303006110). \u003cstrong\u003e(B)\u003c/strong\u003e Deep sequencing of PLN R14del variant carrier transcriptomes to identify SNP co-occuring with PLN c.40_42del. \u003cstrong\u003e(C)\u003c/strong\u003e Exploration of mammalian PLN genomic locus within the adjacent exons of the surrounding CEP85L gene, based on a 45kb alignment. From top to bottom are shown: genomic organisation, homology, regulatory regions, as examined in Grotz 2022, coverage, with grey referring to sequence and white representing sequence lacking. Repetitive elements have been removed from the alignmnet and are indicated by triangles with their length indicated. Highly conserved regions, refering to proposed enhancer regions and area covering exon 1 are enboxed in orange while the upper end of exon 2, including the PLN coding sequence is indicated in blue.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/ffed0f2f542312ffad6054ab.png"},{"id":92252197,"identity":"37e89260-fefe-424c-9166-684b56a4805a","added_by":"auto","created_at":"2025-09-26 10:52:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":664464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePLN transformation process and allelic constellations.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eTransformative strategy to modify the pig PLN locus (upper panel) into a modified allele comprising a removable cassette constituting a human PLN exon 2 expressing WT PLN and a positivie selection cassette, as well as another human PLN exon 2 expressing the R14del variant (middle panel). Triangles: loxP sites. Dotted lines: rs12198461 and rs1051429. Asterisk: R14del modification. Pin: SNP variant differentiating huPLN-WT and huPLN-R14del cassette. After Cre-lox mediated excision, the cassette carrying the huPLNN-R14del exon 2 variant is activatied (lower panel). \u003cstrong\u003e(B)\u003c/strong\u003e Genetic constellations arising from the genetic modification strategy. Homozyous PLNpoWT/poWT serve as control while the heterozygous PLNhuWT/poWT constellation constitute healthy breeding animals and the heterozygous PLNhuR14del/poWT give first evidence of the action of human mutated PLN in pig cardiomyocytes. By combinatorial breeding, the allelic PLNhuR14del/huWT reconsttitute the entire dynamics of human PLN-R14del disease in the pig heart. A homozygous combination of PLNhuR14del/huR14del is expected to lead to an excelerated phenotype, similar to the pathologies in PLN mice (Eijgenraam 2021).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/41294e41ab1b4b90823e200d.png"},{"id":92252189,"identity":"a5c7f715-6975-4095-a201-dd77a342616b","added_by":"auto","created_at":"2025-09-26 10:52:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4341503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic mofication elements. (A)\u003c/strong\u003eThe modification element (vectormod) was created within 2 plasmid cloning steps, involving 2 synthesized elements and a neo-expressing cassette, and used to transform BAC CH242-318M5 (BACWT) into a targeting vector (BACmod), providing extended arms of homology. \u003cstrong\u003e(B)\u003c/strong\u003e RE fingerprinting indicated overall integrity and the exchange of an 11406nt by 8753nt and 8419nt fragments in B23, B24 (asterisks), compared to original BAC (ctr), while B22, B25 indicated imperfect modification. \u003cstrong\u003e(C)\u003c/strong\u003e Sanger sequencing confirmed correct transition between key elements in modified BAC clones. Positions and primers are indicated. Key elements are marked. Electropherograms of reversely orientated primers were transformed in silico for better orientation. Mixed pattern appears in the transitions from huPLN intron (p1, reverse, upper right) into the lox sites, due to the appearance of a GG and a GGG segment (asterisk), the appearance and lack of the R14 codon AGA (bold) in the PLN coding region (p2 forward, middle left) and the transition from the 3´-UTR (p3 forward, middle right) into the neo and the huPLN-R14del cassettes. The palindromic lox sites compromise sequencing quality (lower left). \u003cstrong\u003e(D)\u003c/strong\u003e gRNA were tested for their efficacy to introduce NHEJ-mediated mutations in PKC. Upper left = representative Sanger electropherogram. Lower left = Calculated frequency of NHEJ variants by Synthego. Right: Summary of NHEJ frequencey with 3 gRNA, sequenced in both orientations.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/3340fd420ed68a0240d42d50.png"},{"id":92252200,"identity":"767ce383-5931-451e-8705-655deae6fb1c","added_by":"auto","created_at":"2025-09-26 10:52:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4157971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and screening of gentically modified SCC. (A)\u003c/strong\u003e Primary cells from a WT pig were nucleofected with genetic modification components and seeded to generate single cell clones after negative selction. SCC are split for yielding genetic material for screening. Validated clones are recovered, with the option to treated with Cre-encoding mRNA before SCNT to activate the huPLN-R14del genotype. \u003cstrong\u003e(B)\u003c/strong\u003e Sequential screening concept of SCC by q-PCR-based LOWA (1), Sanger sequencing of PCR spanning the gRNA cutting site (2) and abundance of the huPLN WT- and R14del cassettes (3a) and transition between huPLN-WT and neo cassettes. \u003cstrong\u003e(C)\u003c/strong\u003e Representative LOWA screening of a set of 40 SCC, using qPCR on OCT4 and NANOG genes as reference. Changes in PLN alleles numbers are indicated by orange boxes. * = loss of 1 pig allel, ** = loss of both pig alleles. \u003cstrong\u003e(D)\u003c/strong\u003e Representative Sanger sequencing of region spanning gRNA binding sites, using naturally abundant (G/A) and (C/T) SNP in the pig targeting locus to confirm the genetic constellation of the remaining pig allele. \u003cstrong\u003e(E)\u003c/strong\u003e NHEJ pattern on the pig PLN gene of candidate SCC, resulting from combined use of PLNg1 and PLNg3 to stimulate HR with the targeting BAC. One of the polymorphic sites is indicated (blue box), verifying the identity of remaining porcine PLN allele. Positions of gRNA are indicated by blue line, with PAM represented as dotted line. \u003cstrong\u003e(F) \u003c/strong\u003eRepresentative Sanger electropherogram of humanized coding regions in candidate SCC amplified by PCR 3a, indicating the abundance of a huPLN-WT and –R14del cassettes (upper panel). Representative Sanger electropherogram confirming correct transition from the huPLN-WT to the neo cassette in PCR 3b (lower panel). \u003cstrong\u003e(G)\u003c/strong\u003e Summary of sequential SCC screening, confirming 8 clones to fulfill selection criteria.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/5942d1971c735035adec9eb4.png"},{"id":92252191,"identity":"118d829f-4bc0-4c3a-94a3-018db0f4ab42","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6106006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and propagation of founder animals.\u003c/strong\u003e SCNT-derived founder animals \u003cstrong\u003e(A)\u003c/strong\u003eand screening strategy \u003cstrong\u003e(B)\u003c/strong\u003e, demonstrating the appearance of huPLN-WT and huPLN-R14del constellations \u003cstrong\u003e(C)\u003c/strong\u003e. Asterisk = animals used to establish breeding population. Pink = isolated PKC were used in re-cloning. Localization of allele-specific primers indicated in (B). \u003cstrong\u003e(D)\u003c/strong\u003e Genetic constellation on poPLN-WT allele in SCC (according to Fig. 4E) after GM and in SCNT-derived founder animals. * = breeding animals, ** = isolated PKC were used in re-cloning. \u003cstrong\u003e(E)\u003c/strong\u003e Representatie electropherograms of Sanger seuencing of the poPLN-WT allele in founder pigs. \u003cstrong\u003e(F)\u003c/strong\u003e Breeding strategy to propagate and examine genetic constellations in PLN animals in F1+ generations \u003cstrong\u003e(G)\u003c/strong\u003e, derived from SCNT-generated founder animals 11810 and 11926 and re-cloned founder animal 11809. Square = male, circles = females. Black = WT. Shaded filling = heterozygous huPLN-WT, monochrome filling = homozygous huPLN-WT, bold lines = huPLN-R14del. Attempts of Cre-mediated activation of the R14del cassette are indicated. \u003cstrong\u003e(H)\u003c/strong\u003e Genotyping strategy to deciper genetic constellations in a PLN breeding. \u003cstrong\u003e(I)\u003c/strong\u003e Representative litter derived from inbreeding the 11810 (huPLN-WT) x 11809 lines (huPLN-R14del). Localization of allele-specific primers indicated in (H). \u003cstrong\u003e(J)\u003c/strong\u003e Segregation of neo cassette elements during out- (left) and inbreeding (right). Relative copy numbers are indicated, compared to NANOG and USH1C reference loci. Positioning of the neo-specific PCF product as in Fig. S4A.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/c3dfd18c9e77e56a079eaad3.png"},{"id":92252190,"identity":"e90ef06c-2df7-4313-a596-0775bcb9da9a","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3918053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivating huPLN-R14del genotype by zygote injection.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Distinct stages of cultivated embryos: cleaved embryos after IVF (left), orcein-stained 2-cell embryo (middle) and day 4 embryos (day 4 with blastocysts marked by asterisks (right). \u003cstrong\u003e(B)\u003c/strong\u003e Pigs derived from micro-injected zygotes. \u003cstrong\u003e(C)\u003c/strong\u003eScreening of in vitro cultivated embryos em1-em24, derived from sperm of a huPLN-WT boar and zygote microinjection Cre-mRNA. B2M and poPLN-WT serve as positive control, huPLN-WT and huPLN-R14del indicate the constitution of the cGM allele. Blastocysts (bl.) derived from parthenogenesis, spiked with varying amounts of genomic DNA from poPLN-WT, huPLN-WT and huPLN-R14del animals. \u003cstrong\u003e(D)\u003c/strong\u003eGenotyping of a MI-derived piglets MP1-MP5, using gDNA derived from skin. \u003cstrong\u003e(E)\u003c/strong\u003eSanger sequencing discriminate huPLN-WT (MP1) from huPLN-R14del (MP4) by a 3xG vs 2xG segment (blue boxes), adjacent to the first loxP site (orange box). \u003cstrong\u003e(F)\u003c/strong\u003eDetermination of poPLN-WT, huPLN-WT, neo and huPLN-R14del copy numbers in genomic DNA from skin and blood from MI-derived piglets MP1-MP5. (C, D, F): Animals with distinct genotypes from the breeding program (Fig. S5B) served as controls.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/523900e8dec81c0b3166c89d.png"},{"id":92252926,"identity":"e6b7d2aa-8864-4c67-ada5-5b8828eab18e","added_by":"auto","created_at":"2025-09-26 11:00:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3611167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-natal activation of huPLN-R14del genotype by AAV2/9-mediated Cre delivery.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e After 3-30 week follow-up, the heart of huPLN-WT/poPLN-WT or huPLN-WT/huPLN-WT animals was systematically sampled into 36 left ventricular and 22 right ventricular pieces. In samples of heterozygous huPLN-WT/poPLN-WT were quantified with specific qPCR at genomic (Fig. S2A) and transcript levels \u003cstrong\u003e(B)\u003c/strong\u003e. \u003cstrong\u003e(C)\u003c/strong\u003eQuantification of huPLN-R14del alleles at genomic and transcript levels of LV6, L14, L17, L28, L34 and RV4, RV10, RV15, RV19, RV21 of heterozygous huPLN-WT/poPLN-WT pigs 13566 (6.0x10^15 vp, no coating), 13570 (8.4x10^15vp, no coating). Genomic copy numbers were compared to NANOG and OCT4 reference sites and trancript copy numbers to ACTB, PPIA, TBP and GAPDH house-keeping gene before normalization to huPLN-R14del levels in 2 huPLN-R14del/poPLN-WT pigs. \u003cstrong\u003e(D)\u003c/strong\u003eQuantification of huPLN-R14del genomic copy numbers was done by densitometry of PCR products, using SapI to discriminate huPLN-WT from huPLN-R14del. \u003cstrong\u003e(E)\u003c/strong\u003eProportion of activated R14del cassettes in huPLN-WT/poPLN-WT animals 12918 and 12919, after receiving 1.0x10^15vp AAV-Cre + 180µg G2-cys. \u003cstrong\u003e(F)\u003c/strong\u003e Copy numbers of vector genomes in the respective heart samples and off-target tissue. \u003cstrong\u003e(G)\u003c/strong\u003e Representative NGS data of humanized PLN transcrips of LV36 in an AAV-Cre treated pig, using amplicons spanning the AGA codon of R14 (asterisk) \u003cstrong\u003e(H)\u003c/strong\u003e. \u003cstrong\u003e(I)\u003c/strong\u003e Proportion of huPLN-WT and huPLN-R14del transcripts in AAV-Cre treated homozygous huPLN-WT/huPLN-WT pigs #356 and #358 (both 1.0x10^16vp + 180µg G2-myoP8). \u003cstrong\u003e(J)\u003c/strong\u003e Virus copy numbers in the respective samples, compared to off-target organs.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/52eb3e00f16dbce29f9d85ee.png"},{"id":92252196,"identity":"cb014df3-b3fb-421a-a3c4-956026b17aab","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2503224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript variants on PLN modified loci.\u003c/strong\u003e Features are highlighted for the poPLN-WT \u003cstrong\u003e(A)\u003c/strong\u003e, huPLN-WT \u003cstrong\u003e(B)\u003c/strong\u003e and huPLN-R14del \u003cstrong\u003e(C)\u003c/strong\u003ealleles. RT-PCR amplifying cDNA spanning poPLN-exon1 and the respective exon 2 cassettes (pinkish arrows). Exon-exon boundaries are consistent between pig PLN and humanized PLN variants (left panels). The amino acid sequence in exon are shown corresponding to their codon pattern from R9 to M20 (right panels). AGA-codons in poPLN-WT and huPLN-WT exons 2 are enboxed in gold. SNP discriminating pig and human PLN exon 2 are enboxed in brown.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/167fce206768374405452dc3.png"},{"id":92252931,"identity":"600a6fbc-8536-4b6b-95ab-3fc9aa0c8fc7","added_by":"auto","created_at":"2025-09-26 11:00:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":28332666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/64a5e475-3ed1-415f-9965-a63bf8ee589c.pdf"},{"id":92252199,"identity":"065ee109-2a6c-40c6-ad9c-07ecb17137e0","added_by":"auto","created_at":"2025-09-26 10:52:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2617248,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1. PLN-SERCA interactions.\u003c/strong\u003eInteracting amino acid positions in the SERCA1 (ATP2A1 gene) and SERCA2 (ATP2A2 gene) proteins are highlighted in mammalian species (upper panel) and correlated to the binding region in PLN, as identified in the structural biology studies of Seidel-ref (Seidel 2008), Gorski-ref (Gorski 2015), Glaves-ref (Glaves 2019) and Primeau-ref (Primeau 2018). The respective human amino acid sequence is shown as reference. The positions in other species are dots, if identical, or indicated, if varying.\u003c/p\u003e","description":"","filename":"FigS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/1af683f33e15135e92c13514.tif"},{"id":92252198,"identity":"469c116f-2c52-49d9-a370-9bcdbb65d7d2","added_by":"auto","created_at":"2025-09-26 10:52:31","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1633740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2. Re-nucleofection of SCC.\u003c/strong\u003e Due to the small amount of available cells in SCC portions, nucleofection was optimized by testing Amaxa/Lonza programs with reduced (5x10\u003csup\u003e5\u003c/sup\u003e) cells. Evaluation was done in fluorescence microscopy 48 hours after nucleofection (upper panel). Programs CA-137 and EH-100 were further examined by flow cytometry (lower left) to quantify nucleofection and in bright field microscopy (lower right) to determine viability.\u0026nbsp;\u003c/p\u003e","description":"","filename":"FigS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/3212f11852646b6cdb87bc41.tif"},{"id":92252188,"identity":"760d22ca-15fd-4002-8ddc-37b9d473f8a3","added_by":"auto","created_at":"2025-09-26 10:52:28","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3069364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3. Sequence details of PLN founder animals.\u003c/strong\u003e Localization and specificity of PCR products on genomic DNA in huPLN-WT/poPLN-WT and huPLN-R14del/poPLN-WT founder animals are indicated (top panel). Representative Sanger electropherograms are shown. Orange: spanning first lox site, indicating the appearance of a 2xG motif in huPLN-R14del (#11809) vs 3xG in huPLN-WT (#11810). Blue: spanning open reading frame, indicating that huPLN-WT #11810 comprise both cassettes (mixed pattern with R14 codon), whereas huPLN-R14del retains only the downstream cassette. Amino acid sequences are indicated in one-letter code. Transition from huPLN-WT to neo cassette (pink) and transition from neo cassette to huPLN-R14del cassette (yellow) only appear in huPLN-WT animal #11810.\u003c/p\u003e","description":"","filename":"FigS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/be1e0c3ab37520eb244cd90c.tif"},{"id":92252193,"identity":"fad96dd3-1ae6-457b-aadc-ae1d5b231c44","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1589474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4. Quantfication of genetic elements during breeding.\u003c/strong\u003e Animals of representative lines in Fig. 5B were analyzed for the respective genetic elements \u003cstrong\u003e(A)\u003c/strong\u003e. Copy number ratios are given for poPLN-WT \u003cstrong\u003e(B)\u003c/strong\u003e, huPLN-WT \u003cstrong\u003e(C)\u003c/strong\u003e, huPLN-R14del \u003cstrong\u003e(D)\u003c/strong\u003e constellations, using NANOG and USH1C as genomic reference loci.\u003c/p\u003e","description":"","filename":"FigS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/ecf3b5c769a2d457e651cd98.tif"},{"id":92252194,"identity":"1f190a4a-7426-4990-8261-45c2231a8eee","added_by":"auto","created_at":"2025-09-26 10:52:29","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":85634,"visible":true,"origin":"","legend":"Supplementary Materials and Methods","description":"","filename":"MMdraft250715.docx","url":"https://assets-eu.researchsquare.com/files/rs-7194258/v1/b5edb9b01e96aa083907d1dc.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nPCG, PRV, EW, NK are holder of a patent on the PLN pig model","formattedTitle":"Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAn increasing number of cardiomyopathies, of either dilated or hypertrophic or arrhythmogenic phenotype, has been demonstrated to be caused by (likely) pathogenic genetic variants. Penetrance, severity and onset of the phenotype often vary in a patient population and patho-physiological mechanisms involve secondary effects that are seemingly unrelated to the immediate genetic defect. Studying diseases such as arrhythmogenic cardiomyopathies in animal models also involve physiological and pathophysiological mechanisms that differ between model species and humans. Although frequently used for ischemic and hypertrophic cardiomyopathy studies, especially the staggering heart rate and the limited regulatory capacity of frequency make it harder to directly translate results from the mouse species to human studies. In accordance, the mouse heart is rarely tweaked into ventricular or atrial fibrillation due to its high resting frequency, although high doses of caffeine and catecholamines may induce ventricular tachycardias.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast, pig hearts offer an appealing alternative. Obviously, depending on age and size, pig hearts can grow to similar or even bigger sizes as human hearts, and share proportion, perfusion and valve mechanics. Though being more vulnerable to arrhythmias, pig hearts are studied well using clinical grade electrophysiologic (EP) catheterization and high-resolution mapping (Moretti et al., 2020). They share the occurrence of ischemia-reperfusion arrhythmias, for which they are even more vulnerable and are susceptible for ventricular tachycardias inflicted by exogenous beating cell transplants (Wulkan et al., 2024). Finally, they succumb to a sudden cardiac death phenotype, when (likely) pathogenic genetic variants are present inflicting areas of low or no-amplitude action potentials in EP maps. Of note, successful treatments preventing structural and intracellular deterioration during the unfolding of a genetic cardiomyopathy also result in electrophysiologic stabilization (Bähr et al., 2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIncreasing value of the pig species even further, the toolbox for genetic modification of the species has expanded dramatically (Boettcher et al., 2022; Holm et al., 2016; Howland et al., 2020; Watson et al., 2016), primarily due to the availability of highly potent CRISPR/Cas-based genome modification tools (Klymiuk et al., 2016; Tanihara et al., 2021; Wu et al., 2018). This allowed for fast engineering, regulation of transgene expression (Jin et al., 2014; Klymiuk et al., 2012a) as well as for establishing complex genetic traits such as the targeting of multiple sites (Anand et al., 2023; Griffith et al., 2025) or the (partial) humanization of disease-relevant genes (Grotz et al., 2022). However, evident and insurmountable biological traits, such as longer reproduction cycles, larger size, maintenance costs, etc, hinder implementation of genetically modified (GM) pigs to a scale as it has been established in mice. This limitation specifically includes conditional GM (cGM) models that allow for switching on genetic function in certain cell types, in defined developmental lineages or at desired time points. Aiming at excision, insertion, inversion or exchange of defined genomic sites, cGM generally involve recombinase enzymes that bind and tie defined recognition sites after their stable integration into a genomic site of interest (Oumard et al., 2006; Turan et al., 2013). Very often, cGM comprise Cre enzymes recognizing lox-segments of 34bp (Kuhn et al., 1995), with alternative use of FLTP-FRT, cphi31-attR or others. In mice, recombinases and their recognition sites are mostly applied as independent GM at distinct genomic localizations, allowing for maximal flexibility and modular combination of cGM systems or multiple modification sites, such as genome-wide gene trapping (Schebelle et al., 2010) or multi-cassette accumulation (Kameyama et al., 2010). Several pig lines involving lox-sites for reporter gene induction (Li et al., 2014a; Li et al., 2014b; Wang et al., 2017) or for oncogene activation (Li et al., 2015; Schook et al., 2015) are available. Although GM pigs expressing Cre-recombinase under PTF1A (Kalla et al., 2021), AQP2 (Luo et al., 2014), VASA (Song et al., 2016), MX1 (Chen et al., 2010) or TYR (Oh et al., 2025) promoters have been presented as well, it is common sense that a concept as has been established in mice, providing systematic supply of a vast variety of Cre-lines at commercial or academic exchange basis cannot be expected for other species. Rather, cGM in pig must rely on alternative strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we present a pig model for genetically inherited arrhythmogenic cardiomyopathy, caused by mutated phospholamban (PLN) by the frequent PLN c.40_42delAGA; p.(R14del) pathogenic variant (Nagel et al., 2012), for which we combined deep design approaches with advanced GM strategies and Cre-mediated conditional activation of the pathogenic variant.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cu\u003eMining for PLN translational prospect\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the suitability of \u003cem\u003eSus scrofa\u003c/em\u003e / pig as a model species to investigate PLN dysfunction, we explored the degree of conservation of PLN and its main interacting partners, the SERCA1 (ATP2A1) and SERCA2 (ATP2A2) Ca\u003csup\u003e2+\u003c/sup\u003e pumps. Among representative mammalian species, SERCA proteins show almost complete identity in the sites that have been identified to interfere with PLN (\u003cstrong\u003eFig. S1\u003c/strong\u003e). The short PLN peptide shows strict conservation of the membrane-spanning domain II and loop domain as well as in the regulatory phosphorylation site Ser16 (S16) and Thr17 (T17) \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. Variations in the regulatory and inhibitory regions of PLN such as p.3D/E, p.7C/H (both domain Ia) and p.27K/N (domain Ib) reflect species-specific components to SERCA-PLN interaction. The consequences of variation for PLN-SERCA interactions have not been examined so far. At transcriptomic level, short- and long-read sequencing in 7 PLN-R14del carriers revealed strict correlation of the PLN c.40_42del variant (rs397516784) with the presumably benign rs12198461 and the rs1051429 single nucleotide variant (SNV) in the 3´-UTR of PLN \u003cstrong\u003e(Fig. 1B)\u003c/strong\u003e. This linkage is in line with the hypothesis that a single founder event figures as the origin for the North-Western European PLN-R14del patients (van der Zwaag et al., 2013). Multi-species alignments of the PLN genomic locus showed a relatively low degree of conservation among mammals, compared to previously assessed genes (Grotz et al., 2022). Substantial homology was restricted to the 2 potential enhancer elements upstream of exon 1, the immediate surroundings of exon 1 and the coding region of PLN in exon 2 \u003cstrong\u003e(Fig.1C)\u003c/strong\u003e. Notably, the large 3´-UTR shows substantial divergence between mammals besides a few conserved elements. While the immediate poly-adenylation sites are highly conserved. The particular appearance of repetitive elements within the 3´-UTR in several species suggested lower degree of conservation. In human, all SNV allocated to the PLN 3´-UTR were classified as of uncertain significance or likely benign. Thus, we conclude that pig SERCA proteins can be fully regulated by human PLN. However, we presume that human PLN may have distinct regulatory properties than porcine PLN. Furthermore, post-transcriptional regulation might be distinct between pig and human PLN transcripts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePLN modification strategy\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTo mimic the action of human wild-type (WT) and defective PLN in a pig model, we proposed that the GM design must integrate (i) the causative mutation itself, (ii) the entire human PLN protein constituents, and (iii) the main post-transcriptional regulatory properties of the extended 3´-UTR. The pathological course, establishing disease from a single allele, suggests (iv) a fallback strategy for the case that the dominant negative disease expression interferes with reproduction capacities. The post-translational switching between mono- and pentamer, presumably involved in disease shaping, raises questions about the interaction between human and pig PLN. To integrate all these considerations, we aimed to create a PLN-R14del pig model that combines intact and mutated human PLN protein and a human-like post-transcriptional regulation with a potential fallback strategy that allows for targeted activation of the R14del SNV. We employed a GM strategy that initially established a double-cassette construct, comprising a removable exon that produces an intact human PLN protein and a downstream exon encoding a human R14del protein upon removal of the human PLN-WT cassette \u003cstrong\u003e(Fig. 2A)\u003c/strong\u003e. Integration of the complete GM into the PLN pig locus was achieved by homologous recombination (HR) while the activation of the PLN-R14del was done by Cre-lox mediated excision of the upstream components. The Cre-lox strategy allowed for the expression of huPLN-WT from the complete construct and the expression of huPLN-R14del from the Cre-modified construct. Combinatorial breeding of the huPLN variants \u003cstrong\u003e(Fig. 2B)\u003c/strong\u003e may be used to constitute the patient-relevant huPLN\u003csup\u003eWT/R14del\u0026nbsp;\u003c/sup\u003egenetic constellation or a homozygous huPLN\u003csup\u003eR14del/R14del\u0026nbsp;\u003c/sup\u003evariant, presumably leading to an accelerated phenotype (Eijgenraam et al., 2020). Building on previous work, we followed a GM protocol that combined a modified bacterial artificial chromosome (BAC) (Klymiuk et al., 2013; Klymiuk et al., 2012b), carrying the desired GM and CRISPR/Cas9-mediated double-strand breaks (DSB) (Vochozkova et al., 2019) to stimulate HR of the modified BAC with the target region. Modified BACs provide the capacity for large GM such as the complete PLN-WT – neo – PLN-R14del modification and extended regions of homology to the targeting region, avoiding the necessity for isogenic homologous arms. To modify the BAC CH242-318M5, covering the pig PLN locus, we combined gene synthesis, sequential plasmid cloning and bacterial recombineering \u003cstrong\u003e(Fig. 3A)\u003c/strong\u003e. Modification of the BAC was confirmed by end-point PCR spanning homologous arms \u003cstrong\u003e(Fig. 3B)\u003c/strong\u003e and integrity of the modified BAC was verified by restriction enzyme finger printing (Auch et al., 2022) \u003cstrong\u003e(Fig. 3C)\u003c/strong\u003e. To induce HR-stimulating DSB, 3 gRNAs were designed and tested, revealing substantially distinct NHEJ mutation rates (PLN-BACg1: 11-12%, PLN-BACg2: 0% and PLN-BACg3: 40-41%) \u003cstrong\u003e(Fig. 3.D)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eHumanizing the pig PLN locus\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAccording to established protocols (Richter 2012) \u003cstrong\u003e(Fig. 4.A)\u003c/strong\u003e, primary cell lines of 2 distinct WT pigs were nucleofected with plasmids expressing Cas9, gRNAs and a linearized modification BAC. After clonal selection and passaging, 161 single cell clones (SCC) were cryo-conserved and screened for homologous recombination in a sequential protocol \u003cstrong\u003e(Fig. 4B)\u003c/strong\u003e. qPCR-based loss-of-wildtype allele (LoWA) detected 100-6000 genomic copies / µl in the DNA preparations of SCC and rejected 138 clones as potentially modified at the 5´-region of the modification \u003cstrong\u003e(Fig. 4.C)\u003c/strong\u003e. One of the 23 candidate SCC did not retain a pig PLN allele, suggesting bi-allelic modification of the target locus. The other candidates were further screened by sequencing a PCR product spanning the CRISPR-gRNA binding site. By assessing 2 naturally occurring SNV in the amplicon as well as the appearance of NHEJ-mediated mutations \u003cstrong\u003e(Fig. 4D)\u003c/strong\u003e, 13 SCC were confirmed to retain a single pig PLN allele. Sanger sequencing of the remaining PLN allele in these SCC confirmed that the majority acquired a gap that exactly referred to the cutting sites of the gRNA used to stimulate HR \u003cstrong\u003e(Fig. 4E)\u003c/strong\u003e. Alternative NHEJ pattern was observed occasionally. Final validation for abundance of the huPLN-WT, huPLN-R14del and neo-coding regions designated a total of 8 candidate clones for SCNT. For SCNT experiments, SCC were recovered and prepared for SCNT, including the nucleofection with Cre-encoding plasmid or lipofection with Cre-mRNA to activate the huPLN-R14del cassette. 4 SCNT were conducted, using pooled SCC preparations, according to (Kurome et al., 2015). 2 litters delivered 3 viable and 2 stillborn piglets \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e. Genotyping by endpoint PCR \u003cstrong\u003e(Fig. 5B, C)\u003c/strong\u003e revealed 3 of them to carry the complete huPLN-WT modification and 2 retaining the huPLN-R14del cassette, indicating Cre-mediated excision after nucleofection with Cre-encoding plasmid. Sanger sequencing of PCR products spanning the modification segment confirmed the abundance and identity of huPLN-WT and huPLN-R14del genetic elements and the correct transition between them \u003cstrong\u003e(Fig. S3)\u003c/strong\u003e. Sequencing of the poPLN-WT allele allowed the correlation of founder animals 11809 as well as the majority of re-cloned animals to SCC_111, while the origin of the others remained unclear due to the consistent NHEJ pattern in the other SCC used in SCNT \u003cstrong\u003e(Fig. 5D, E)\u003c/strong\u003e. To further explore the genomic constellation of GM elements, animals #11810, #11926 and #12435, a viable clone of #11809, were raised and propagated by a breeding scheme that combines outbreeding for increased fertility and fitness with inbreeding to achieve a huPLN\u003csup\u003eWT/R14del\u0026nbsp;\u003c/sup\u003egenetic constellation at the PLN locus \u003cstrong\u003e(Fig. 5F, G)\u003c/strong\u003e. Quantitative exploration for the poPLN-WT, huPLN-WT, huPLN-R14del \u003cstrong\u003e(Fig. S4)\u003c/strong\u003e genetic elements confirmed genotyping by endpoint PCR \u003cstrong\u003e(Fig. 5H, I)\u003c/strong\u003e, with genotype proportions according to the Mendelian rules of inheritance. Quantification of the neo selection cassette in F1 animals \u003cstrong\u003e(Fig. 5J)\u003c/strong\u003e revealed that the neo element segregated from the PLN-locus in the #11809 lineage and purified PLN-R14del individuals lacking the neo element were selected for further breeding. In line #11926, multiple copies of neo fragments were inherited with the PLN-locus, suggesting an inappropriate genetic modification of the target locus and clearing of this line from further breeding attempts. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eActivating of huPLN-R14del by mRNA micro-injection\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eIn parallel to the cleaning of the PLN-R14del line #11809 from the neo fragment by Mendelian segregation, we aimed at an alternative activation protocol of huPLN-R14del in the pure PLN-WT line #11810, using the Cre-mediated activation in GM zygotes. Sperm of a huPLN-WT F1 boar was used to mate synchronized WT sows of the Libechov minipig breed. Next day, sows were slaughtered, and presumptive zygotes were flushed from oviduct, cultivated in improved defined medium and microinjected with highly pure mRNA carrying a codon-optimized variant of Cre-recombinase, according to published data (Whitworth et al., 2018). To test recombination efficiency, microinjected zygotes were cultured \u003cem\u003ein vitro\u003c/em\u003e (\u003cstrong\u003eFig. 6A\u003c/strong\u003e) for 4-5 days. The developmental potential of embryos was not compromised by micro-injection as 58.33% of embryos reached morula or blastocyst stage. After termination, 24 embryos at varying stages were used for nested end-point PCR. While the pattern of two PCR products aiming at B2M reference site and pig PLN locus was not fully conclusive, PCR sensitive for the complete modification encoding huPLN-WT or modified cassette encoding huPLN-R14del indicated partial efficacy of Cre-mediated cassette excision after micronjection \u003cstrong\u003e(Fig. 6B)\u003c/strong\u003e. Next, micro-injected zygotes were cultivated for one day and then approx. 30 embryos at the 2-4 cell stage were transferred to synchronized gilts. Out of 3 embryo transfers, 1 pregnancy was established, delivering 5 viable offspring \u003cstrong\u003e(Fig. 6C, D)\u003c/strong\u003e. Genotyping revealed 1 piglet to be poPLN\u003csup\u003eWT/WT\u003c/sup\u003e, while 2 piglets carried the non-modified huPLN-WT cassette and 2 others were successfully modified to express the huPLN-R14del cassette. To test mosaicism in MI-derived piglets, Sanger sequencing of PCR products spanning across the first loxP site revealed that piglets MP3 and MP4 had a pattern that unambiguously correlated to the huPLN-R14del genotype, without any sign of remaining huPLN-WT \u003cstrong\u003e(Fig. 6E)\u003c/strong\u003e. Likewise, qPCR-based copy number determination on gDNA from skin and blood of the MI-derived piglets revealed a clear huPLN-WT pattern in MP1 and MP5 whereas MP3 and MP4 were huPLN-R14del without any indication of a remaining huPLN-WT pattern \u003cstrong\u003e(Fig. 6F)\u003c/strong\u003e. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePost-natal activation of huPLN-R14del genotype\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eIn a third attempt, we sought to activate the huPLN-R14del cassette in a viable animal. For this, F1 piglets of heterozygous huPLN-WT/poPLNWT or homozygous huPLN-WT/huPLN-WT genotypes injected intravenously at an age of 4-8 weeks with 1.0x10\u003csup\u003e15\u003c/sup\u003e-1.0x10\u003csup\u003e16\u003c/sup\u003e vp of Cre-encoding AAV2/9, with or without PAMAM coating or improved cardiomyocyte transduction. Heart tissue was systematically sampled for the left (LV) and right ventricles (RV) \u003cstrong\u003e(Fig. 7A)\u003c/strong\u003e and examined for induction of the huPLN-R14del cassette and virus abundance. One pair of heterozygous animals was kept for 21 weeks injection of AAV without coating. At genomic levels, the Cre efficacy ranged between 0.6-3.8% in representative LV and 0.4-2.1% in representative RV samples, compared to the levels in huPLN-R14del/poPLN-WT animals, whereas the abundance of huPLN-R14del transcripts reached 9.7-27.9% for LV and 0.9-13.4% for RV \u003cstrong\u003e(Fig. 7B, C)\u003c/strong\u003e. In an attempt with G2-cys PAMAM coated AAV, another pair of animals indicated increased activation of huPLN-R14del cassettes, correlating with evident amounts of virus genomes in heart tissue samples 3 weeks after administration \u003cstrong\u003e(Fig. 7D-F)\u003c/strong\u003e. Coating with G2-myoP8 PAMAM resulted in varying efficacy in homozygous huPLN-WT/huPLN-WT pigs. In one animal, the huPLN-R14del variant made up 16-37% of PLN transcripts, while in another animal the value only reached 2%, whereas the amount of virus genomes was comparable in both animals. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eTranscriptional constellations at modified PLN loci\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTo validate correct expression of the respective genotypes, transcription of poPLN-WT, huPLN-WT and huPLN-R14del was determined by Sanger sequencing of RT-PCR amplicons spanning poPLN exon 1 and the respective downstream exons. In poPLN\u003csup\u003eWT/WT\u0026nbsp;\u003c/sup\u003eanimals, the exon boundaries and coding sequence \u003cstrong\u003e(Fig. 8A)\u003c/strong\u003e were in line with GenBank entry, confirming conserved splice donor and splice acceptor sites in multi-species alignment \u003cstrong\u003e(Fig. 1C)\u003c/strong\u003e. In huPLN-WT constellation, poPLN-WT exon 1 was predominantly spliced to huPLN-WT exon 2. At low frequency (\u0026lt;5%) alternative splicing occurred to the downstream huPLN-R14del exon \u003cstrong\u003e(Fig. 8B)\u003c/strong\u003e. In huPLN-R14del constellation, the splicing from poPLN-WT exon 1 to huPLN-R14del exon 2 was effective, confirming that splice acceptor sites acted correctly when shuttled between species \u003cstrong\u003e(Fig. 8C)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we combine CRISPR-based homologous recombination with Cre-mediated recombinase-mediated cassette excision (RMCE) to establish a complex humanized knock-in pig model for PLN-mediated heart disease. While some of the GM components have been presented by us and others in independent studies earlier, true cGM approaches have not yet been presented before in a systematic manner.\u003c/p\u003e\n\u003cp\u003eIn pigs, various GM methods have been tested (Hamze et al., 2025). In the pre-CRISPR era, site-directed modifications were only achieved for a few designated loci, such as GGTA (reviewed in (Klymiuk et al., 2010)), CFTR (Klymiuk et al., 2012b; Rogers et al., 2008) or DMD (Klymiuk et al., 2013). Nowadays they are often established by direct injection of CRISPR/Cas components into zygotes (Fu et al., 2024; Geisert et al., 2023; Maynard et al., 2021; Whitworth et al., 2017), either aiming at the error-prone NHEJ pathway or homology-directed repair (HDR), using single strand deoxy-nucleotides (ssODN) to introduce a defined mutation (Klymiuk et al., 2016). Larger site-directed modifications, however, still appear challenging and are commonly established and verified in primary cells (Vochozkova et al., 2019), before they are used as nucleus donor in SCNT (Kurome et al., 2015). Most large GM attempts build on targeting vectors that flank the desired GM with homologous arms of several 100bp up to few kb lengths, whereas we have gained vast experience in manipulating BACs for becoming targeting vectors. Initially created to clone segments of up to 200kb eukaryotic DNA into plasmid vectors during genome sequencing projects, BACs represent a resource that can be modified extensively by bacterial recombineering in dedicated E.coli strains (Auch et al., 2022), giving rise to vector with huge genomic regions that serve as homologous arms during recombination with the target region in pig primary cells (Beshr et al., 2017; Klymiuk et al., 2013; Klymiuk et al., 2012b). While chromatin accessibility is commonly thought as critical for GM efficacy (Chari et al., 2015; Jensen et al., 2017), combinations of BAC vectors with CRISPR/Cas-mediated DSB performed well on target sites that were considered silent in pig primary cells, such as USH1C (Grotz et al., 2022) or PLN, as we demonstrated here.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContrary to the widely used technologies for constitutive GM in defined genomic sites, methods that allow for cGM remain sparse in pigs, not to speak of dissemination as it has been seen in mouse. Initially, Cre-mediated cassette excision of genetic modifications (Li et al., 2014a; Moon et al., 2012) demonstrated proof-of-concept in pig cells in vitro, but only few reports demonstrated RMCE in vivo (Moretti et al., 2020; Schook et al., 2015). The generation of numerous pig strains expressing Cre under certain conditions (Chen et al., 2010; Kalla et al., 2021; Luo et al., 2014; Oh et al., 2025; Song et al., 2016) suggests that the reason behind this is not merely scientific. Indeed, distribution or share of existing GM pig lines between research groups has been rather confined so far, and we see little perspective for an exchange system that facilitates crossbreeding GM strains with Cre-lines upon request in a foreseeable future. With advancing biomedical models in pig, however, cGM approaches will become more relevant, as we exemplify here for a model for PLN-R14del arrhythmogenic cardiomyopathy. The chosen strategy involved several considerations: While aberrant phosphorylation of the mutated protein alone enhance SERCA inhibition by direct interaction (Vafiadaki et al., 2022; Vostrikov et al., 2015), the assembly of PLN into pentamers is highly relevant for its regulatory properties (Funk et al., 2023; Glaves et al., 2019; Smeazzetto et al., 2017; Traaseth et al., 2007; Wittmann et al., 2015)), suggesting direct interaction of WT and mutated PLN proteins. For the difference in pig and human PLN, the option to combine human PLN-R14del with human PLN-WT was therefore seen as precondition. Further, the dominant negative pathophysiology raised concerns about detrimental effects on reproduction, in case of early disease onset, suggesting a cGM approach as fallback strategy. Finally, SCNT of primary cells remains a bottleneck technology in biomedical engineering, as it critically depends on restricted and seasonal cloning capacities, on the very source of primary cells or the developmental capacities of validated single cell clones (Kurome et al., 2013; Richter et al., 2012). The introduction of a complex but single cGM into one allele of the target locus in primary cells by gene editing and its subsequent modulation meticulously balanced risk with effort in our GM pipeline. The combination of gene synthesis and plasmid cloning allowed for efficient construction of a 9kb modifications construct and its efficient integration into a BAC covering the pig PLN gene (Fig. 3). Human PLN-WT was expressed from the constitutive stage of the GM while Cre-mediated RMCE effectively induced the R14del genotype (Fig. 7). Once established, human WT and R14del variants of the modified PLN gene were combined by crossbreeding to precisely re-constitute the dynamics between human PLN-WT and R14del in mono- and oligomers (Fig. 2B). Further, the comparison of distinct Cre application strategies to activate the PLN-R14del cassette illustrated the capacities to establish cGM in pig without the necessity to create a complementary GM line expressing Cre as transgene.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe treatment of verified SCC with Cre-encoding mRNA, via lipofection, or plasmid nucleofection prior to SCNT elegantly established both, huPLN-WT and huPLN-R14del genetic variants within a single cloning experiment (Fig. 5C). Success rates of the SCNT procedure and the number of viable founder piglets are remarkable, recapitulating similar efficacy with the same (Grotz et al., 2022) and other primary cell lines (Stirm et al., 2023). The thriving re-cloning capacities of primary cells from a deceased PLN-R14del founder individual (#11809) further confirmed the used primary cell line as highly potent for GM and SCNT. Indeed, extended genotyping (Fig. 5H, S2) demonstrated that several cell clones out of the verified SCC pool developed to founder animals. Together, SCNT is a powerful tool to establish complex GM in pig and combination of gene editing and Cre-recombinase facilitates the generation of constitutive and activated variants of cGM in one go, if all components of primary cell manipulation, in vitro reproductive technologies and embryo transfer were properly executed. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDirect micro-injection of Cre-mRNA into the cytoplasm of cultivated embryos that were produced with sperm of a boar containing the entire cGM cassette (Fig. 6), represented a valuable alternative to SCNT of Cre-treated primary cells. In contrast to SCNT, which facilitates the generation of genetically identical clones from a thoroughly characterized primary cell population, IVF-derived pigs resemble littermates because of germ lines meiosis. Thus, parental alleles are inherited in a ratio of 1:1 and only 50% offspring carry a desired genetic trait, if this appeared in a mono-allelic constellation in one of the parents. From a pragmatic point of view, this genetic drawback is often outcompeted by the beneficial reproductive features as IVF appears technically more robust and yields higher developmental potential than SCNT embryos (Isom et al., 2012; Yamanaka et al., 2009; Zhai et al., 2022), correlating to larger litter sizes and, importantly, better vitality of the piglets even after embryo micromanipulation (Chen et al., 2022). In our approach, we further stimulated the developmental potential by flushing embryos from the oviducts of inseminated sows, avoiding the lower developmental competence associated to the use of in vitro produced embryos (Bauer et al., 2010; Cambra et al., 2021; Chen et al., 2022). Relevantly, the manipulation of zygotes is often associated with mosaicism, i.e. the activity of the injected compound after the first cleavage of the embryo, resulting in distinct genetic constellations in the cells of an organism. This problem has been seen to a particular extent after injecting CRISPR/Cas components into mouse (Hashimoto et al., 2016), bovine (Wei et al., 2022) or pig zygotes (Navarro-Serna et al., 2021; Whitworth et al., 2017). The completely different mode of action of Cre recombinase and its much faster kinetics, however minimized this risk in mouse zygote injection (Araki et al., 1995; Luckow et al., 2009), which is in line with the unambiguous huPLN-WT and huPLN-R14del genotypes in both blastocysts and piglets derived by micro-injection (Fig. 6E, F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, post-natal activation or the R14del cassette revealed significant cGM efficiencies by intravenous application of a Cre-encoding AAV into young piglets. At first sight, the discrepancies between relatively low Cre-efficacy at genomic and much higher activation at transcript level appear puzzling. Considering, however, that cardiomyocytes were the preferred target of AAV2/9 vectors and likewise also the sole source of PLN transcripts in the heart, transcriptional assessment provides true evidence for the desired cGM activation events. Assuming that there was no regulatory interaction between huPLN-WT and huPLN-R14del alleles, the quantification of PLN transcripts provides additional insight: Any increase in R14del expression indicates a nuclear event and allows for the precise quantification of Cre-mediated events, independent of the nuclear status of cardiomyocytes. While combining Cre-application with reproduction technologies provides a “clean” genotype in the whole organism, the mosaic cGM pattern after post-natal Cre-application, provides the perspective for studying spatial effects, in particular if application can be done at high concentrations during restricted local administration (Lampela et al., 2024). Given the often focal development of cardiomyopathies, this approach may provide fundamentally new insight into disease-forming events and the pathophysiological mechanisms behind their dissemination across larger areas of the heart. Evidently, post-natal application remains the sole option for cGM activation in case of embryonal or fetal fatal genetic disorders.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, we present a comprehensive design of a cGM pig model for arrhythmogenic cardiomyopathies and document effective ways of cGM activation by applying Cre recombinase at distinct developmental stages. Our findings pave the way for novel future cGM approaches in pig but also in other species for which the complementary transgenic Cre-driving lines are lacking and their development, maintenance and distribution is challenging. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAll procedures involving human patients and animals were approved and supervised by the respective local authorities. Detailed Methods are described in a supplementary document.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePRV\u0026nbsp;\u003c/strong\u003econtributed to the design of the model, performed genetic manipulation of pig genome and contributed to the characterization of animals. \u003cstrong\u003eMV\u003c/strong\u003e contributed to zygote microinjection. \u003cstrong\u003eJMC\u003c/strong\u003e designed and directed and mainly performed characterization of animals and embryos. \u003cstrong\u003eAB\u003c/strong\u003e designed and mainly preformed animal intervention and directed breeding program. \u003cstrong\u003eEMM\u003c/strong\u003e contributed to animal intervention, characterization of animals and breeding program. \u003cstrong\u003eMK\u003c/strong\u003e contributed to SCNT and provided parthenogenic embryos. \u003cstrong\u003eVZ\u003c/strong\u003e, \u003cstrong\u003eTG\u003c/strong\u003e contributed to SCNT. \u003cstrong\u003eBK\u003c/strong\u003e contributed to SCNT and performed embryo transfer. \u003cstrong\u003eMLC\u003c/strong\u003e and \u003cstrong\u003eHCH\u003c/strong\u003e contributed to animal intervention. \u003cstrong\u003eTB\u003c/strong\u003e, \u003cstrong\u003eVR\u003c/strong\u003e, \u003cstrong\u003eIML\u003c/strong\u003e provided AAV and explored transduction efficacy. \u003cstrong\u003ePvT\u003c/strong\u003e, \u003cstrong\u003eMH\u003c/strong\u003e explored patient material. \u003cstrong\u003eSJ\u003c/strong\u003e and \u003cstrong\u003eJJ\u003c/strong\u003e performed embryo transfer. \u003cstrong\u003eCGCG\u003c/strong\u003e contributed to animal characterization and breeding management. \u003cstrong\u003eZE, JM\u003c/strong\u003e contributed to breeding management, directed embryo flushing and contributed to the design of the manuscript. \u003cstrong\u003ePAD\u003c/strong\u003e, \u003cstrong\u003ePCG\u003c/strong\u003e contributed to the design of the model. \u003cstrong\u003eDD\u003c/strong\u003e directed and contributed to zygote microinjection. \u003cstrong\u003eEW\u003c/strong\u003e contributed to the design of the model and provided SCNT technology. \u003cstrong\u003eCK\u003c/strong\u003e contributed to the design of the model and to the writing of the manuscript. \u003cstrong\u003eNK\u003c/strong\u003e led overall work, design of the model, characterization and writing of the manuscript. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnand, R.P., Layer, J.V., Heja, D., Hirose, T., Lassiter, G., Firl, D.J., Paragas, V.B., Akkad, A., Chhangawala, S., Colvin, R.B.\u003cem\u003e, et al.\u003c/em\u003e (2023). Design and testing of a humanized porcine donor for xenotransplantation. Nature\u003cem\u003e 622\u003c/em\u003e, 393-401.\u003c/li\u003e\n\u003cli\u003eAraki, K., Araki, M., Miyazaki, J., and Vassalli, P. (1995). Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A\u003cem\u003e 92\u003c/em\u003e, 160-164.\u003c/li\u003e\n\u003cli\u003eAuch, H., Klymiuk, N., and Runa-Vochozkova, P. (2022). Modifying Bacterial Artificial Chromosomes for Extended Genome Modification. Methods Mol Biol\u003cem\u003e 2495\u003c/em\u003e, 67-90.\u003c/li\u003e\n\u003cli\u003eB\u0026auml;hr, A., Hoppmann, P., Bozoglu, T., Stirm, M., Luksch, I., Ziegler, T., Hornaschewitz, N., Shrestha, S., Shashikadze, B., St\u0026ouml;ckl, J.\u003cem\u003e, et al.\u003c/em\u003e (2023). Gene Therapy for Cardiomyopathy associated with Duchenne Muscular Dystrophy in a Pig Model. bioRxiv, 2023.2010.2002.560452.\u003c/li\u003e\n\u003cli\u003eBauer, B.K., Isom, S.C., Spate, L.D., Whitworth, K.M., Spollen, W.G., Blake, S.M., Springer, G.K., Murphy, C.N., and Prather, R.S. (2010). Transcriptional profiling by deep sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos. Biol Reprod\u003cem\u003e 83\u003c/em\u003e, 791-798.\u003c/li\u003e\n\u003cli\u003eBeshr, G., Sikandar, A., Jemiller, E.M., Klymiuk, N., Hauck, D., Wagner, S., Wolf, E., Koehnke, J., and Titz, A. (2017). Photorhabdus luminescens lectin A (PllA): A new probe for detecting alpha-galactoside-terminating glycoconjugates. J Biol Chem\u003cem\u003e 292\u003c/em\u003e, 19935-19951.\u003c/li\u003e\n\u003cli\u003eBoettcher, A.N., Schachtschneider, K.M., Schook, L.B., and Tuggle, C.K. (2022). Swine models for translational oncological research: an evolving landscape and regulatory considerations. Mamm Genome\u003cem\u003e 33\u003c/em\u003e, 230-240.\u003c/li\u003e\n\u003cli\u003eCambra, J.M., Martinez, E.A., Rodriguez-Martinez, H., Gil, M.A., and Cuello, C. (2021). Transcriptional Profiling of Porcine Blastocysts Produced In Vitro in a Chemically Defined Culture Medium. Animals (Basel)\u003cem\u003e 11\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eChari, R., Mali, P., Moosburner, M., and Church, G.M. (2015). Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods\u003cem\u003e 12\u003c/em\u003e, 823-826.\u003c/li\u003e\n\u003cli\u003eChen, L., Li, L., Pang, D., Li, Z., Wang, T., Zhang, M., Song, N., Yan, S., Lai, L.X., and Ouyang, H. (2010). Construction of transgenic swine with induced expression of Cre recombinase. Animal\u003cem\u003e 4\u003c/em\u003e, 767-771.\u003c/li\u003e\n\u003cli\u003eChen, P.R., Uh, K., Redel, B.K., Reese, E.D., Prather, R.S., and Lee, K. (2022). Production of Pigs From Porcine Embryos Generated in vitro. Frontiers in Animal Science\u003cem\u003e Volume 3 - 2022\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eEijgenraam, T.R., Boukens, B.J., Boogerd, C.J., Schouten, E.M., van de Kolk, C.W.A., Stege, N.M., Te Rijdt, W.P., Hoorntje, E.T., van der Zwaag, P.A., van Rooij, E.\u003cem\u003e, et al.\u003c/em\u003e (2020). The phospholamban p.(Arg14del) pathogenic variant leads to cardiomyopathy with heart failure and is unreponsive to standard heart failure therapy. Sci Rep\u003cem\u003e 10\u003c/em\u003e, 9819.\u003c/li\u003e\n\u003cli\u003eFu, B., Ma, H., Huo, X., Zhu, Y., and Liu, D. (2024). CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications. Biomolecules\u003cem\u003e 14\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eFunk, F., Kronenbitter, A., Hackert, K., Oebbeke, M., Klebe, G., Barth, M., Koch, D., and Schmitt, J.P. (2023). Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation. Cardiovasc Res\u003cem\u003e 119\u003c/em\u003e, 1568-1582.\u003c/li\u003e\n\u003cli\u003eGeisert, R.D., Johns, D.N., Pfeiffer, C.A., Sullivan, R.M., Lucas, C.G., Simintiras, C.A., Redel, B.K., Wells, K.D., Spencer, T.E., and Prather, R.S. (2023). Gene editing provides a tool to investigate genes involved in reproduction of pigs. Mol Reprod Dev\u003cem\u003e 90\u003c/em\u003e, 459-468.\u003c/li\u003e\n\u003cli\u003eGlaves, J.P., Primeau, J.O., Espinoza-Fonseca, L.M., Lemieux, M.J., and Young, H.S. (2019). The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA. Biophys J\u003cem\u003e 116\u003c/em\u003e, 633-647.\u003c/li\u003e\n\u003cli\u003eGriffith, B.P., Grazioli, A., Singh, A.K., Tully, A., Galindo, J., Saharia, K.K., Shah, A., Strauss, E.R., Odonkor, P.N., Williams, B.\u003cem\u003e, et al.\u003c/em\u003e (2025). Transplantation of a genetically modified porcine heart into a live human. Nat Med\u003cem\u003e 31\u003c/em\u003e, 589-598.\u003c/li\u003e\n\u003cli\u003eGrotz, S., Schafer, J., Wunderlich, K.A., Ellederova, Z., Auch, H., Bahr, A., Runa-Vochozkova, P., Fadl, J., Arnold, V., Ardan, T.\u003cem\u003e, et al.\u003c/em\u003e (2022). Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes. EMBO Mol Med\u003cem\u003e 14\u003c/em\u003e, e14817.\u003c/li\u003e\n\u003cli\u003eHamze, J.G., Cambra, J.M., Navarro-Serna, S., and Martinez-Serrano, C.A. (2025). Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives. Genomics\u003cem\u003e 117\u003c/em\u003e, 111014.\u003c/li\u003e\n\u003cli\u003eHashimoto, M., Yamashita, Y., and Takemoto, T. (2016). Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol\u003cem\u003e 418\u003c/em\u003e, 1-9.\u003c/li\u003e\n\u003cli\u003eHolm, I.E., Alstrup, A.K., and Luo, Y. (2016). Genetically modified pig models for neurodegenerative disorders. J Pathol\u003cem\u003e 238\u003c/em\u003e, 267-287.\u003c/li\u003e\n\u003cli\u003eHowland, D., Ellederova, Z., Aronin, N., Fernau, D., Gallagher, J., Taylor, A., Hennebold, J., Weiss, A.R., Gray-Edwards, H., and McBride, J. (2020). Large Animal Models of Huntington\u0026apos;s Disease: What We Have Learned and Where We Need to Go Next. J Huntingtons Dis\u003cem\u003e 9\u003c/em\u003e, 201-216.\u003c/li\u003e\n\u003cli\u003eIsom, S.C., Li, R.F., Whitworth, K.M., and Prather, R.S. (2012). Timing of first embryonic cleavage is a positive indicator of the in vitro developmental potential of porcine embryos derived from in vitro fertilization, somatic cell nuclear transfer and parthenogenesis. Mol Reprod Dev\u003cem\u003e 79\u003c/em\u003e, 197-207.\u003c/li\u003e\n\u003cli\u003eJensen, K.T., Floe, L., Petersen, T.S., Huang, J., Xu, F., Bolund, L., Luo, Y., and Lin, L. (2017). Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency. FEBS Lett\u003cem\u003e 591\u003c/em\u003e, 1892-1901.\u003c/li\u003e\n\u003cli\u003eJin, Y.X., Jeon, Y., Lee, S.H., Kwon, M.S., Kim, T., Cui, X.S., Hyun, S.H., and Kim, N.H. (2014). Production of pigs expressing a transgene under the control of a tetracycline-inducible system. PLoS One\u003cem\u003e 9\u003c/em\u003e, e86146.\u003c/li\u003e\n\u003cli\u003eKalla, D., Flisikowski, K., Yang, K., Sanguesa, L.B., Kurome, M., Kessler, B., Zakhartchenko, V., Wolf, E., Lickert, H., Saur, D.\u003cem\u003e, et al.\u003c/em\u003e (2021). The Missing Link: Cre Pigs for Cancer Research. Front Oncol\u003cem\u003e 11\u003c/em\u003e, 755746.\u003c/li\u003e\n\u003cli\u003eKameyama, Y., Kawabe, Y., Ito, A., and Kamihira, M. (2010). An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange. Biotechnol Bioeng\u003cem\u003e 105\u003c/em\u003e, 1106-1114.\u003c/li\u003e\n\u003cli\u003eKlymiuk, N., Aigner, B., Brem, G., and Wolf, E. (2010). Genetic modification of pigs as organ donors for xenotransplantation. Mol Reprod Dev\u003cem\u003e 77\u003c/em\u003e, 209-221.\u003c/li\u003e\n\u003cli\u003eKlymiuk, N., Blutke, A., Graf, A., Krause, S., Burkhardt, K., Wuensch, A., Krebs, S., Kessler, B., Zakhartchenko, V., Kurome, M.\u003cem\u003e, et al.\u003c/em\u003e (2013). Dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. Hum Mol Genet\u003cem\u003e 22\u003c/em\u003e, 4368-4382.\u003c/li\u003e\n\u003cli\u003eKlymiuk, N., Bocker, W., Schonitzer, V., Bahr, A., Radic, T., Frohlich, T., Wunsch, A., Kessler, B., Kurome, M., Schilling, E.\u003cem\u003e, et al.\u003c/em\u003e (2012a). First inducible transgene expression in porcine large animal models. FASEB J\u003cem\u003e 26\u003c/em\u003e, 1086-1099.\u003c/li\u003e\n\u003cli\u003eKlymiuk, N., Mundhenk, L., Kraehe, K., Wuensch, A., Plog, S., Emrich, D., Langenmayer, M.C., Stehr, M., Holzinger, A., Kroner, C.\u003cem\u003e, et al.\u003c/em\u003e (2012b). Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl)\u003cem\u003e 90\u003c/em\u003e, 597-608.\u003c/li\u003e\n\u003cli\u003eKlymiuk, N., Seeliger, F., Bohlooly, Y.M., Blutke, A., Rudmann, D.G., and Wolf, E. (2016). Tailored Pig Models for Preclinical Efficacy and Safety Testing of Targeted Therapies. Toxicol Pathol\u003cem\u003e 44\u003c/em\u003e, 346-357.\u003c/li\u003e\n\u003cli\u003eKuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995). Inducible gene targeting in mice. Science\u003cem\u003e 269\u003c/em\u003e, 1427-1429.\u003c/li\u003e\n\u003cli\u003eKurome, M., Geistlinger, L., Kessler, B., Zakhartchenko, V., Klymiuk, N., Wuensch, A., Richter, A., Baehr, A., Kraehe, K., Burkhardt, K.\u003cem\u003e, et al.\u003c/em\u003e (2013). Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set. BMC Biotechnol\u003cem\u003e 13\u003c/em\u003e, 43.\u003c/li\u003e\n\u003cli\u003eKurome, M., Kessler, B., Wuensch, A., Nagashima, H., and Wolf, E. (2015). Nuclear transfer and transgenesis in the pig. Methods Mol Biol\u003cem\u003e 1222\u003c/em\u003e, 37-59.\u003c/li\u003e\n\u003cli\u003eLampela, J., Pajula, J., Jarvelainen, N., Siimes, S., Laham-Karam, N., Kivela, A., Mushimiyimana, I., Nurro, J., Hartikainen, J., and Yla-Herttuala, S. (2024). Caridac vein retroinjections provide an efficient approach for global left ventricular gene transfer with adenovirus and adeno-associated virus. Sci Rep\u003cem\u003e 14\u003c/em\u003e, 1467.\u003c/li\u003e\n\u003cli\u003eLi, S., Edlinger, M., Saalfrank, A., Flisikowski, K., Tschukes, A., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A.\u003cem\u003e, et al.\u003c/em\u003e (2015). Viable pigs with a conditionally-activated oncogenic KRAS mutation. Transgenic Res\u003cem\u003e 24\u003c/em\u003e, 509-517.\u003c/li\u003e\n\u003cli\u003eLi, S., Flisikowska, T., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A., Wolf, E., Flisikowski, K., and Schnieke, A. (2014a). Dual fluorescent reporter pig for Cre recombination: transgene placement at the ROSA26 locus. PLoS One\u003cem\u003e 9\u003c/em\u003e, e102455.\u003c/li\u003e\n\u003cli\u003eLi, X., Yang, Y., Bu, L., Guo, X., Tang, C., Song, J., Fan, N., Zhao, B., Ouyang, Z., Liu, Z.\u003cem\u003e, et al.\u003c/em\u003e (2014b). Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res\u003cem\u003e 24\u003c/em\u003e, 501-504.\u003c/li\u003e\n\u003cli\u003eLuckow, B., Hanggli, A., Maier, H., Chilla, S., Loewe, R.P., Dehmel, S., Schlondorff, D., Loetscher, P., Zerwes, H.G., and Muller, M. (2009). Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice. Genesis\u003cem\u003e 47\u003c/em\u003e, 545-558.\u003c/li\u003e\n\u003cli\u003eLuo, W., Li, Z., Huang, Y., Han, Y., Yao, C., Duan, X., Ouyang, H., and Li, L. (2014). Generation of AQP2-Cre transgenic mini-pigs specifically expressing Cre recombinase in kidney collecting duct cells. Transgenic Res\u003cem\u003e 23\u003c/em\u003e, 365-375.\u003c/li\u003e\n\u003cli\u003eMaynard, L.H., Humbert, O., Peterson, C.W., and Kiem, H.P. (2021). Genome editing in large animal models. Mol Ther\u003cem\u003e 29\u003c/em\u003e, 3140-3152.\u003c/li\u003e\n\u003cli\u003eMoon, J., Kim, S., Park, H., Kang, J., Park, S., Koo, O., da Torre, B.R., Saadeldin, I.M., Lee, B., and Jang, G. (2012). Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre-loxP. Zygote\u003cem\u003e 20\u003c/em\u003e, 423-425.\u003c/li\u003e\n\u003cli\u003eMoretti, A., Fonteyne, L., Giesert, F., Hoppmann, P., Meier, A.B., Bozoglu, T., Baehr, A., Schneider, C.M., Sinnecker, D., Klett, K.\u003cem\u003e, et al.\u003c/em\u003e (2020). Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nat Med\u003cem\u003e 26\u003c/em\u003e, 207-214.\u003c/li\u003e\n\u003cli\u003eNagel, B., Gruber-Sedlmayr, U., Uhrig, S., Stollberger, C., Klopocki, E., and Finsterer, J. (2012). Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants. BMC Med Genet\u003cem\u003e 13\u003c/em\u003e, 60.\u003c/li\u003e\n\u003cli\u003eNavarro-Serna, S., Hachem, A., Canha-Gouveia, A., Hanbashi, A., Garrappa, G., Lopes, J.S., Paris-Oller, E., Sarrias-Gil, L., Flores-Flores, C., Bassett, A.\u003cem\u003e, et al.\u003c/em\u003e (2021). Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination. CRISPR J\u003cem\u003e 4\u003c/em\u003e, 132-146.\u003c/li\u003e\n\u003cli\u003eOh, D., Hong, N., Eun, K., Lee, J., Cai, L., Kim, M., Choi, H., Jawad, A., Ham, J., Park, M.G.\u003cem\u003e, et al.\u003c/em\u003e (2025). Generation of a genetically engineered porcine melanoma model featuring oncogenic control through conditional Cre recombination. Sci Rep\u003cem\u003e 15\u003c/em\u003e, 1616.\u003c/li\u003e\n\u003cli\u003eOumard, A., Qiao, J., Jostock, T., Li, J., and Bode, J. (2006). Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology. Cytotechnology\u003cem\u003e 50\u003c/em\u003e, 93-108.\u003c/li\u003e\n\u003cli\u003eRichter, A., Kurome, M., Kessler, B., Zakhartchenko, V., Klymiuk, N., Nagashima, H., Wolf, E., and Wuensch, A. (2012). Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol\u003cem\u003e 12\u003c/em\u003e, 84.\u003c/li\u003e\n\u003cli\u003eRogers, C.S., Stoltz, D.A., Meyerholz, D.K., Ostedgaard, L.S., Rokhlina, T., Taft, P.J., Rogan, M.P., Pezzulo, A.A., Karp, P.H., Itani, O.A.\u003cem\u003e, et al.\u003c/em\u003e (2008). Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science\u003cem\u003e 321\u003c/em\u003e, 1837-1841.\u003c/li\u003e\n\u003cli\u003eSchebelle, L., Wolf, C., Stribl, C., Javaheri, T., Schnutgen, F., Ettinger, A., Ivics, Z., Hansen, J., Ruiz, P., von Melchner, H.\u003cem\u003e, et al.\u003c/em\u003e (2010). Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps. Nucleic Acids Res\u003cem\u003e 38\u003c/em\u003e, e106.\u003c/li\u003e\n\u003cli\u003eSchook, L.B., Collares, T.V., Hu, W., Liang, Y., Rodrigues, F.M., Rund, L.A., Schachtschneider, K.M., Seixas, F.K., Singh, K., Wells, K.D.\u003cem\u003e, et al.\u003c/em\u003e (2015). A Genetic Porcine Model of Cancer. PLoS One\u003cem\u003e 10\u003c/em\u003e, e0128864.\u003c/li\u003e\n\u003cli\u003eSmeazzetto, S., Armanious, G.P., Moncelli, M.R., Bak, J.J., Lemieux, M.J., Young, H.S., and Tadini-Buoninsegni, F. (2017). Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA. J Biol Chem\u003cem\u003e 292\u003c/em\u003e, 21330-21339.\u003c/li\u003e\n\u003cli\u003eSong, Y., Lai, L., Li, L., Huang, Y., Wang, A., Tang, X., Pang, D., Li, Z., and Ouyang, H. (2016). Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig. FEBS Open Bio\u003cem\u003e 6\u003c/em\u003e, 50-55.\u003c/li\u003e\n\u003cli\u003eStirm, M., Shashikadze, B., Blutke, A., Kemter, E., Lange, A., Stockl, J.B., Jaudas, F., Laane, L., Kurome, M., Kessler, B.\u003cem\u003e, et al.\u003c/em\u003e (2023). Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52. Proc Natl Acad Sci U S A\u003cem\u003e 120\u003c/em\u003e, e2301250120.\u003c/li\u003e\n\u003cli\u003eTanihara, F., Hirata, M., and Otoi, T. (2021). Current status of the application of gene editing in pigs. J Reprod Dev\u003cem\u003e 67\u003c/em\u003e, 177-187.\u003c/li\u003e\n\u003cli\u003eTraaseth, N.J., Verardi, R., Torgersen, K.D., Karim, C.B., Thomas, D.D., and Veglia, G. (2007). Spectroscopic validation of the pentameric structure of phospholamban. Proc Natl Acad Sci U S A\u003cem\u003e 104\u003c/em\u003e, 14676-14681.\u003c/li\u003e\n\u003cli\u003eTuran, S., Zehe, C., Kuehle, J., Qiao, J., and Bode, J. (2013). Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. Gene\u003cem\u003e 515\u003c/em\u003e, 1-27.\u003c/li\u003e\n\u003cli\u003eVafiadaki, E., Haghighi, K., Arvanitis, D.A., Kranias, E.G., and Sanoudou, D. (2022). Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca(2+) Handling and Arrhythmogenesis. Int J Mol Sci\u003cem\u003e 23\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003evan der Zwaag, P.A., van Rijsingen, I.A., de Ruiter, R., Nannenberg, E.A., Groeneweg, J.A., Post, J.G., Hauer, R.N., van Gelder, I.C., van den Berg, M.P., van der Harst, P.\u003cem\u003e, et al.\u003c/em\u003e (2013). Recurrent and founder mutations in the Netherlands-Phospholamban p.Arg14del mutation causes arrhythmogenic cardiomyopathy. Neth Heart J\u003cem\u003e 21\u003c/em\u003e, 286-293.\u003c/li\u003e\n\u003cli\u003eVochozkova, P., Simmet, K., Jemiller, E.M., Wunsch, A., and Klymiuk, N. (2019). Gene Editing in Primary Cells of Cattle and Pig. Methods Mol Biol\u003cem\u003e 1961\u003c/em\u003e, 271-289.\u003c/li\u003e\n\u003cli\u003eVostrikov, V.V., Soller, K.J., Ha, K.N., Gopinath, T., and Veglia, G. (2015). Effects of naturally occurring arginine 14 deletion on phospholamban conformational dynamics and membrane interactions. Biochim Biophys Acta\u003cem\u003e 1848\u003c/em\u003e, 315-322.\u003c/li\u003e\n\u003cli\u003eWang, K., Jin, Q., Ruan, D., Yang, Y., Liu, Q., Wu, H., Zhou, Z., Ouyang, Z., Liu, Z., Zhao, Y.\u003cem\u003e, et al.\u003c/em\u003e (2017). Cre-dependent Cas9-expressing pigs enable efficient in vivo genome editing. Genome Res\u003cem\u003e 27\u003c/em\u003e, 2061-2071.\u003c/li\u003e\n\u003cli\u003eWatson, A.L., Carlson, D.F., Largaespada, D.A., Hackett, P.B., and Fahrenkrug, S.C. (2016). Engineered Swine Models of Cancer. Front Genet\u003cem\u003e 7\u003c/em\u003e, 78.\u003c/li\u003e\n\u003cli\u003eWei, J., Brophy, B., Cole, S.A., Moormann, J., Boch, J., and Laible, G. (2022). Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos. Front Genet\u003cem\u003e 13\u003c/em\u003e, 925913.\u003c/li\u003e\n\u003cli\u003eWhitworth, K.M., Benne, J.A., Spate, L.D., Murphy, S.L., Samuel, M.S., Murphy, C.N., Richt, J.A., Walters, E., Prather, R.S., and Wells, K.D. (2017). Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs. Transgenic Res\u003cem\u003e 26\u003c/em\u003e, 97-107.\u003c/li\u003e\n\u003cli\u003eWhitworth, K.M., Cecil, R., Benne, J.A., Redel, B.K., Spate, L.D., Samuel, M.S., Prather, R.S., and Wells, K.D. (2018). Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs. Transgenic Res\u003cem\u003e 27\u003c/em\u003e, 167-178.\u003c/li\u003e\n\u003cli\u003eWittmann, T., Lohse, M.J., and Schmitt, J.P. (2015). Phospholamban pentamers attenuate PKA-dependent phosphorylation of monomers. J Mol Cell Cardiol\u003cem\u003e 80\u003c/em\u003e, 90-97.\u003c/li\u003e\n\u003cli\u003eWu, H., Liu, Q., Shi, H., Xie, J., Zhang, Q., Ouyang, Z., Li, N., Yang, Y., Liu, Z., Zhao, Y.\u003cem\u003e, et al.\u003c/em\u003e (2018). Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci\u003cem\u003e 75\u003c/em\u003e, 3593-3607.\u003c/li\u003e\n\u003cli\u003eWulkan, F., Romagnuolo, R., Qiang, B., Valdman Sadikov, T., Kim, K.P., Quesnel, E., Jiang, W., Andharia, N., Weyers, J.J., Ghugre, N.R.\u003cem\u003e, et al.\u003c/em\u003e (2024). Stem cell-derived cardiomyocytes expressing a dominant negative pacemaker HCN4 channel do not reduce the risk of graft-related arrhythmias. Front Cardiovasc Med\u003cem\u003e 11\u003c/em\u003e, 1374881.\u003c/li\u003e\n\u003cli\u003eYamanaka, K., Sugimura, S., Wakai, T., Kawahara, M., and Sato, E. (2009). Difference in sensitivity to culture condition between in vitro fertilized and somatic cell nuclear transfer embryos in pigs. J Reprod Dev\u003cem\u003e 55\u003c/em\u003e, 299-304.\u003c/li\u003e\n\u003cli\u003eZhai, Y., Yu, H., An, X., Zhang, Z., Zhang, M., Zhang, S., Li, Q., and Li, Z. (2022). Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation - a comparative analysis between early porcine embryos and their counterparts in other three mammalian species. BMC Genomics\u003cem\u003e 23\u003c/em\u003e, 772.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7194258/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7194258/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Complex diseases such as progressive cardiomyopathies are often insufficiently reflected in small animal and in vitro models. Large animal species such as pigs provide a valuable alternative, but constitutive genetic manipulation (cGM) has not yet been applied to pig in an effective manner, mainly due to biological and logistic limitations. Here, we describe the generation of a novel humanized model for PLN-mediated cardiomyopathy and compare different methods of activating a pathogenic R14del mutation by Cre-mediated recombination. Both, the Cre- treatment of pig primary cells before somatic cell nuclear transfer as well as the micro-injection of Cre-encoding mRNA into zygotes, were similarly efficient in delivering piglets with an activated R14del mutation. Alternatively, administration of Cre-encoding AAV into piglets was sufficient in cGM, albeit at to a varying extent. Together, we here describe a highly effective process to establish complex cGM traits in pig and demonstrate that the lack of Cre-driver lines can be compensated by various interventations during reproduction or post-natally.","manuscriptTitle":"Activating a patient-relevant mutation of a genetically defined heart disease in a humanized pig model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 10:52:23","doi":"10.21203/rs.3.rs-7194258/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"lab-animal","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"laban","sideBox":"Learn more about [Lab Animal](http://www.nature.com/laban/)","snPcode":"","submissionUrl":"","title":"Lab Animal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a69619a2-1f6d-4512-953c-ef507d2b5578","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55331079,"name":"Biological sciences/Biotechnology/Animal biotechnology/Genetic engineering"},{"id":55331080,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2026-02-09T18:36:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 10:52:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7194258","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7194258","identity":"rs-7194258","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}