Validation and Optimization of Breeding Strategy for miR-141/200c Knockout Mice to Eliminate Off-Target Gene Silencing using FLPo Deleter

Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele View ORCID Profile Sanjeev Kumar Yadav , View ORCID Profile Rashmi Srivastava , Mary-Katherine Cormier , Katie Lowther , Siu-Pok Yee , Rajkumar Verma doi: https://doi.org/10.1101/2025.09.02.673774 Sanjeev Kumar Yadav 1 Department of Neurosciences , UConn Health Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sanjeev Kumar Yadav Rashmi Srivastava 1 Department of Neurosciences , UConn Health Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rashmi Srivastava Mary-Katherine Cormier 1 Department of Neurosciences , UConn Health Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katie Lowther 2 Center for Mouse Genome Modification , UConn Health, Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Siu-Pok Yee 2 Center for Mouse Genome Modification , UConn Health, Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: raverma{at}uchc.edu syee{at}uchc.edu Rajkumar Verma 1 Department of Neurosciences , UConn Health Farmington CT 06030 Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: raverma{at}uchc.edu syee{at}uchc.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT MicroRNAs (miRNAs) of the miR-200 family specifically miR-141 and miR-200c regulate neurogenesis, differentiation, and epithelial mesenchymal transitions in development. Dysregulation of these miRNAs is associated with several diseases including cancer and stroke. The Mirc13tm1Mtm/Mmjax mouse line, which targets the miR-141/200c cluster, was originally generated and described by Park et al. 2012 as a knockout-first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line. To harness the full potential of this mouse line, it would require a two-step breeding process: breed with FLP mice to excise lacZ/neo cassettes followed by breeding with Cre to delete the floxed miRNA cluster( Park, et al. 2012 ). However, many studies either bypassed removal of the lacZ/Neo cassettes and treated the mouse line as Mirc13 knockouts or bred directly with Cre mouse lines, which could lead to unpredictable recombination and genotypes. In this study, we show that the presence of the Neo cassette influences expression of neighboring genes. We demonstrate that it is essential to follow the two-step breeding plan. INTRODUCTION microRNAs (miRNAs) are small, noncoding RNAs (∼22 nucleotides) that regulate gene expression post-transcriptionally and play pivotal roles in development and disease. Originally discovered in C. elegans( Lee, et al. 1993 ; Wightman, et al. 1993 ) and later shown to be evolutionarily conserved across species( Pasquinelli, et al. 2000 ), miRNAs are now recognized as critical regulators in cardiovascular( Small, et al. 2010 ; van Rooij and Kauppinen 2014 ), neurological( Wu, et al. 2016 ; Verma, et al. 2018 ), and oncogenic pathways( Humphries and Yang 2015 ; Jin, et al. 2017 ), holding significant potential for therapeutic applications( Janssen, et al. 2013 ; Verma, et al. 2018 ) While cell-based studies have elucidated many of these mechanisms, in vivo models remain essential to fully understand the functions of miRNAs under physiological and pathological contexts. Park et al. established a key genetic resource for in vivo miRNA investigation by generating conditional knockout-first mouse lines for conserved miRNAs, including the miR-141/200c cluster (Mirc13tm1Mtm/Mmjax)( Park, et al. 2012 ). This cluster, located on mouse chromosome 6, encodes two members of the miR-200 family implicated in neurogenesis( Choi, et al. 2008 ; Beclin, et al. 2016 ), differentiation( Zhang, et al. 2015 ), epithelial–mesenchymal transition( Tseng, et al. 2017 ), cancer( Chen, et al. 2013 ; Jin, et al. 2017 ), stroke( Small, et al. 2010 ; Yadav, et al. 2024 ), and Alzheimer’s disease( Wu, et al. 2016 ). To make miR-141/200c cluster KO mice, Park et al. used a targeting vector to incorporate a lacZ reporter and β-actin-neo selection cassette flanked by FRT sites and loxP sites surrounding the miR-141/200c cluster. This enabled a two-step recombination strategy: first, FLP-mediated removal of the lacZ/neo cassette generates a clean conditional (floxed) allele, and then Cre-mediated excision removes the miR-141/200c cluster( Park, et al. 2012 ). However, many subsequent studies bypassed the essential FLP recombination step( Guan, et al. 2018 ; Hoefert, et al. 2018 ; Ji, et al. 2018 ; Liu, et al. 2018 ; Wu, et al. 2019 ; Xu, et al. 2025 ). In such cases, the lacZ/neo selection cassette remains in the targeted allele, which can transcriptionally silence neighboring genes( Testa, et al. 2004 ; Raja, et al. 2024 ) or alter local expression patterns due to the strong β-actin promoter driving the cassette( Pham, et al. 1996 ), thereby confounding phenotypic interpretation. Other studies attempted cassette removal using FLPe deleter strains; however, several commonly used FLPe lines exhibit variable recombination efficiency, which may lead to incomplete or mosaic excision depending on the deleter strain and genomic target locus( Tran, et al. 2017 ; Mostofa, et al. 2022 ; Tran, et al. 2024 ). Consequently, a small proportion of animals may retain the selection cassette despite FLPe breeding, resulting in heterogeneous genotypes within experimental cohorts. Several studies have reported that residual selection cassettes can unintentionally suppress the expression of nearby genes( Olson, et al. 1996 ; Pham, et al. 1996 ; Skarnes, et al. 2011 ). In the case of the miR-141/200c cluster, genes located in close proximity—including Ptpn6, Phb2, Atn1, Eno2, and Eng1 ( www.Ensembl.org )—may be affected. Because these genes are known to play important roles in neuronal function and mitochondrial integrity( Whitton, et al. 1993 ; Bernabeu, et al. 2009 ; Xu, et al. 2014 ; Isgrò, et al. 2015 ; He, et al. 2023 ), unintended suppression could lead to misleading interpretations or the apparent loss of phenotype in knockout mice generated without prior removal of the neo cassette. To overcome these limitations, we optimized the original “knockout-first” design suggested by Park et al. 2012( Park, et al. 2012 ) by employing high-efficiency FLPo deleter mice instead of FLPe or β-actin-FLP strains( Raymond and Soriano 2007 ; Wu, et al. 2009 ; Kranz, et al. 2010 ). FLPo recombinase exhibits greater thermostability and recombination efficiency in mammalian cells( Raymond and Soriano 2007 ; Wu, et al. 2009 ; Kranz, et al. 2010 ), ensuring complete excision of the lacZ/neo cassette and restoration of an unperturbed floxed allele before Cre-mediated deletion. This refinement minimizes off-target silencing, preserves adjacent gene expression, and produces genetically clean global or tissue-specific miR-141/200c knockouts. Here, we present a validated, optimized, and detailed breeding strategy using FLPo deleter mice that ensures complete removal of the lacZ/neo cassette and precise deletion of the miR-141/200c cluster. This work establishes a robust framework for generating unambiguous cell-specific or conditional knockouts of the miR-141/200c cluster, facilitating accurate studies of miRNA function in neural and systemic disease models. MATERIAL AND METHODS Mouse Strains All mouse strains used in this study were obtained from the Jackson Laboratory (Bar Harbor, ME): Mirc13 tm1Mtm /Mmjax line (MMRRC stock #34657( Park, et al. 2012 )) (hereon referred as Mirc13 tm1 line); B6.129S4-Gt(ROSA)26Sor tFLP )Sor/J (ROSA-FLPo; Jax# 012930)( Raymond and Soriano 2007 ); 129S1/Sv-Hprt1 tm1(CAG-cre)Mnn /J) (Hprt-Cre; Jax# 004302)( Tang, et al. 2002 ) and B6.129P2-Lyz2 tm1(cre)Ifo /J) (LysMCre; Jax # 004781)( Clausen, et al. 1999 ). All mice were maintained under standard conditions at ambient temperature and humidity, with ad libitum access to pellet food and water. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Connecticut Health Center. Genotyping of Mouse Strains Tail biopsies (∼4 mm) were collected in DNase-free microtubes and incubated in 50 ml of alkali buffer (25mM NaOH and 0.2mM EDTA) at 100°C for one hour and an equal volume of neutralization buffer (40mM Tris buffer, pH 7.5) was added. One microliter of the DNA was used for PCR genotyping with 2× JumpStart™ Taq ReadyMix (Sigma-Aldrich P2893). Primer pairs are listed in Table 1 . PCR was performed according to the manufacturer’s instructions. PCR products were fractionated on 2% agarose gels prepared in 1× Tris-Acetate-EDTA (TAE) buffer (Bio-Rad 1610743) with SYBR™ Safe DNA stain (Invitrogen S33102) and visualized under UV illumination. View this table: View inline View popup Download powerpoint Table 1. List of different sets of primer sequences (with amplicon size) used in PCR amplification β-Galactosidase (lacZ) Staining A β-galactosidase staining kit (Sigma-Aldrich GALS-1KT) was used to assess lacZ reporter expression. Mice were deeply anesthetized with Avertin (250 mg/kg, i.p.), perfused with ice-cold PBS, and brains were collected for cryosections (10 µm, Superfrost Plus slides). Sections were fixed with fixation solution (provided with kit) for 10 min, and LacZ expression was determined using a β-galactosidase staining kit according to manufacturer’s instructions. Slides were mounted in 70% glycerol and imaged under bright-field microscopy. RNA Isolation, cDNA Synthesis, and Real-Time PCR Total RNA was extracted from the olfactory bulb, frontal and parietal cortex, hippocampus, cerebellum, heart, lung, and liver using TRIzol or miRVana kits (Ambion Life Technologies, 15596018, AM1560). cDNA synthesis for miRNAs and mRNAs was performed using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher 4366596) and iScript cDNA Synthesis Kit (Bio-Rad 1708891), respectively. TaqMan assays included miR-141-3p (000463), miR-200c-3p (002300), U6snRNA (001973), Ptpn6 (Mm00469153_m1), Phb2 (Mm00476104_m1), Atn1 (Mm00492256_m1), Eno2 (Mm00469062_m1), Emg1 (Mm00496596_m1), and Gapdh (Mm99999915_g1) (Thermo Fisher Scientific). PCR was performed using TaqMan Universal PCR Master Mix II (No UNG; Thermo Fisher 4440040). Relative expression was determined by the 2^–ΔΔCt method using U6 and Gapdh as normalizers for miRNA and mRNA targets, respectively. RNA Fluorescence In Situ Hybridization (RNA-FISH) RNA-FISH was performed using Molecular Instruments protocols. Fixed tissue sections were pre-hybridized at 37 °C and incubated overnight with 16 nM of the miR-141-3p probe. Following stringent SSCT washes, signal amplification was performed using snap-cooled hairpins (h1/h2; 6 pmol each) incubated overnight at room temperature. Sections were mounted with antifade DAPI UltraCruz Mounting Solution (Santa Cruz SC-24941) and imaged by fluorescence microscopy. Statistical Analysis Data are presented as mean ± SD. Relative expression values derived from the 2^–ΔΔCt method were used for statistical analysis. Statistical analyses were performed using GraphPad Prism (San Diego, CA). Comparisons between two groups were conducted using a two-tailed unpaired Student’s t -test, while multiple group comparisons were analyzed using one-way ANOVA followed by Bonferroni post hoc correction. Data distribution was assessed for normality prior to applying parametric tests. A p value < 0.05 was considered statistically significant. Blinding was implemented during data acquisition and analysis where feasible. Each dot In graphs represent an individual mouse. RESULTS Genomic Organization of the Mirc13 Mouse Lines and Genotyping of the Mouse Lines The Mirc13 tm1Mtm /Mmjax line (MMRRC stock #34657) mouse line, hereon referred as Mirc13 tm1 line, available from the Jax MMRCC is a knockout-first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line. The miR-141/200c (Mirc13) cluster is located within intron 1 of the lncRNA, GM15884 . As shown in Fig 1A , the targeted allele contains an engrailed 2 (En2) splice acceptor followed by an internal ribosomal entry site (ires) sequence coupled to lacZ reporter sequence and polyadenylation signal sequence. This mouse line was originally generated by gene targeting in embryonic stem (ES) cells via homologous recombination. A β-actin-Neo cassette was placed 3’ of the lacZ polyA sequence in the targeting vector to confer G418 selection to help screen for targeted ES cell clones. The Neo cassette and the lacZ reporter was separated by a single loxP site. The lacZ/Neo sequences were flanked by a pair of Frt sites and inserted into intron 1 of GM15884 5’ of the floxed Mirc13 cluster. With the polyadenylation sequence placed 3’ of the lacZ sequence, it will terminate any GM15884 3’-downstream transcription, including the Mirc13 cluster, therefore, this mouse line is a double knockout of both GM15884 and Mirc13 cluster (knockout first). We first performed PCR genotyping of Mirc13 tm1/+ pups using primer pairs as shown in Table 1 . As shown in Fig 2A , we detected amplicon of 340 bp and 432 bp specific to the Mirc13 WT and floxed allele, respectively. We were also able to amplify fragments of 315 bp and 280 bp specific to lacZ and Neo, respectively, confirming the genotype of the Mirc13 tm1/+ mice we obtained from MMRCC/Jax. We then bred the Mirc13 tm1/+ pups together to obtain Mirc13 tm1/tm1 pups for expression analyses as described below. PCR genotyping only detected the 432 bp fragment specific to the Mirc13 floxed allele as well as the lacZ and neo specific amplicons. The absence of the Mirc13 WT specific amplicon of 340 bp confirmed WT that these were homozygous Mirc13 tm1 mice ( Fig 2A ). Download figure Open in new tab Fig. 1 Genomic organization of Mirc13tm1 allele and after Flpo and Cre mediated recombination. A The Mirc13tm1 mouse line was originally designed as a a knockout first, reporter-tagged insertion with conditional potential (conditional-ready) mouse line for Mirc13 (miR-141/200c cluster). Insertion of the en-lacZ gene trap cassette with prevent any subsequent 3’-downstream transcription leading to the lack of GM15884 and Mirc13 expression. B When these mice were bred with Rosa26-Flpo, Flpo will facilitate excision of the LacZ/Neo cassette to generate the Mirc13fl allele with wildtype level of expression of both GM15884 and mir141/200c. C When Mirc13fl mice were bred with Cre, Cre will mediate excision of the Mirc13 cluster sequence to generate Mirc13-allele with wildtype GM15884 expression and the lack of miR141/200c expression. Download figure Open in new tab Fig. 2 PCR Genotyping of mice with different Mirc13 alleles. A Generation of Mirc13 tm1/tm1 mice by breeding Mirc13 tm1/+ mice, which contain lacZ/Neo cassette and floxed Mirc13 sequence , together; B Removal of lacZ and Neo cassette by breeding Mirc13 tm1/+ mice with Rosa26 FLPo/+ mice. Positive Mirc13 fl/+ mice, lacking the lacZ/Neo cassette (315 bp amplicon), were marked by a red box; C Generation of heterozygous Mirc13 +/− (KO) mice by breeding Mirc13 fl/fl with Hprt CAG-Cre/+ mice. Positive Mirc13 +/− mice, lacking a 432 bp amplicon specific to the Mirc1fl 1/+ allele, were marked by a red box; D Heterozygous Mirc13 +/− mice were bred together to generate homozygous Mirc13 −/− global homozygous KO mice. These mice were identified by the presence of a KO-specific amplicon (352 bp) and the loss of wt specific amplicon (340 bp) and were marked by the red box. PCR were performed using primer pairs as shown in Table 1 and amplicons specific for lacZ is 315 bp, Mirc + is 340 bp, Mirc13 fl is 432 bp, Mirc13 - is 352 bp, Rosa26 is 603 bp, Flpo is 300 bp and Cre is 100 bp. As described above, the lacZ/Neo sequences are flanked by a pair of Frt sites, which can be excised by Flp recombinase resulting in the generation of functional Mirc13 conditional knockout (conditional later) mice expressing both GM15884 and miR-141/200c as shown in Fig 1B . To accomplish this, we bred Mirc13 tm1/+ mice with Rosa26 Flpo/+ mice (Jax #012930). As shown in Fig 2B , genotyping results showed the absence of lacZ specific amplicon of 315 bp (lane 1 and 2) thereby confirming successful excision of the lacZ/Neo cassette and generation of Mirc13 fl/+ pups. To generate Mirc13 knockout (KO) mice, we bred Mirc13 fl/+ pups with Hprt CAG-Cre/+ mice (Fig1C). To detect the knockout allele, a forward primer (mir5Fn3) was designed to target the region upstream of the frt/loxP site and was paired with the Jax 11312 reverse primer downstream of the 3’ loxP site. This primer pair will generate two amplicons, one specific to the WT allele (1.3 kb) and one specific to the knockout allele that is smaller in size due to Cre-mediated recombination (353 bp). As shown in Fig 2C , PCR genotyping showed that we were able to amplify the predicted fragments of 1.3 kb (lane 1) or 352 bp (lanes 2 and 3) specific to Mirc13 WT and KO alleles, respectively. We anticipated that we should obtain either Mirc13 +/+ or Mirc13 +/− pups from this breeding pair. However, we did not detect both amplicons in heterozygous Mirc13 +/− mice. This is most likely because our PCR conditions favors amplification of the smaller amplicon specific to the knockout allele, and the pups shown in Fig 2C lanes 2 and 3, were Mirc13 +/− mice. We further bred Mirc13 +/− mice together and mice were genotyped using the same primers described above for the knockout allele and also with the original Jax primer pairs specific to the 3’ loxP site in order to detect the presence or absence of the WT allele. We were able to obtain Mirc13 −/− mice as shown by PCR genotyping that was positive only for the KO-specific amplicon and no WT-specific amplicon was observed ( Fig 2D , lane 2). The other littermates were either Mirc13 +/− , which were positive for both amplicons ( Fig 2D lanes 1 and 4) or Mirc13 +/+ as revealed by the presence of the WT-specific amplicon only ( Fig 2D lane 3). Expression Analysis of Homozygous Mirc13 tm1 Mice The En2 splice acceptor is designed to facilitate splicing between exon 1 of GM15884 with the ires-lacZ reporter sequence. Thus, the targeted allele will generate a fusion transcript consisting of exon 1 of the disrupted endogenous GM15884 , a lncRNA, ires and lacZ coding sequence. PCR genotyping confirmed presence of the lacZ reporter ( Fig 2A ). Since the ires-lacZ gene trap cassette is inserted into intron 1 of the endogenous GM15884 and placed under the GM15884 transcriptional regulatory sequence, lacZ expression will faithfully recapitulate spatiotemporal expression of the endogenous GM15884 LncRNA, which should also reveal the spatiotemporal expression of the miR-141/200c cluster (Mirc13) located within intron 1 of GM15884 . However, the polyadenylation sequence placed 3’ with respect to the lacZ sequence will prevent further 3’-downstream transcription, including the Mirc13 cluster, to accomplish the knockout first task. This mouse line is practically a double knockout of both GM15884 and Mirc13 cluster. We first examined lacZ expression, which should reveal endogenous expression of GM15884 and miR-141-3p and miR-200c-3p, by x-gal staining of b-galactosidase, the gene product of lacZ. Strong b-galactosidase activity was detected in the olfactory bulb, with weaker staining near the lung–tracheal junction, and minimal expression in the heart and liver ( Fig. 3A ). To further characterize expression of Mirc13, we isolated total RNA from the brain olfactory bulb and lungs of Mirc13 tm1/tm1 and Mirc13 +/+ mice and performed RT-qPCR to examine the level of miR-141-3p and miR-200c-3p expression. Our results showed that miR-141-3p and miR-200c-3p were highly expressed in the olfactory bulb and lung (ΔCT 10) in Mirc13 +/+ mice, consistent with the miRNA expression atlas( Rishik, et al. 2025 ) ( Fig. 3B and C ). A significant and near-complete reduction of miR-141-3p ( Fig 3D &E and miR-200c-3p ( Fig. 3F & G) expression was observed in the olfactory bulb and lungs in Mirc13 tm1/tm1 mice. Data are presented as mean ± SD (*p < 0.05 vs. WT control). Download figure Open in new tab Fig. 3 Expression analyses of Mirc13 tm1/tm1 mice. A . LacZ expression as revealed by x–gal staining of olfactory bulb (OB) and lung (LG); B , C Basal expression of miR-141-3p and miR-200c-3p was analyzed by RT-qPCR in olfactory bulb, lung, heart and liver of wildtype mice. Relative expressions of miR-141-3p ( D &E ) and miR-200c-3p ( F & G ) were assessed by RT-qPCR in the brain olfactory bulb and lungs of Mirc13 tm1/tm1 KO mice compared to (Mirc13 +/+ ) littermate control mice. Data are presented as mean ± SD (*p < 0.05 vs. Mirc13 +/+ littermate control). Blue column denotes Mirc13 +/+ and yellow column denotes Mirc13 tm1/tm1 . Presence of β-actin-Neo cassette leads to Transcriptional Interference of Neighboring Genes The Mirc13 tm1 mouse line was originally generated by gene targeting in embryonic stem cells via homologous recombination. A lacZ polyA sequence was included in the targeting vector to confer G418 positive selection to screen for targeted ES cell clones. However, the Neo cassette containing a strong ubiquitous β-actin promoter was not removed prior to generation of the Mirc13 tm1 mouse line. It has been reported that insertion of a strong promoter within a gene would not only disrupt endogenous expression, but it could also potentially lead to dysregulation of neighboring genes and resulted in unanticipated phenotypic alterations( Olson, et al. 1996 ; Pham, et al. 1996 ; Skarnes, et al. 2011 ). To further investigate the influence of the b-actin-Neo cassette on neighboring gene expression, we identified 5 genes, Ptpn6, Phb2, Atn1, Eno2 and Emg1 that are important for brain function, located within 50 kb from Mirc13. Total RNA was isolated from olfactory bulb of Mirc13 +/+ and Mirc13 tm1/tm1 mice and the levels of neighboring gene expression were characterized by RT-qPCR. As shown in Fig 4A-C , expression of Ptpn6, Phb2, and Atn1 was significantly decreased (±>1.5 folds threshold, p<0.05) in Mirc13 tm1/tm1 mice as compared to Mirc13 +/+ mice, while Eno2 and Emg1 showed a similar decreasing trend ( Fig 4D and E ). In Mirc13 −/− mice, the expression Ptpn6, Phb2, Atn1, Eno2, and Emg1 did not show significantly change (±>1.5 folds threshold, p<0.05) as compared to Mirc13 fl/fl mice ( Fig 4F to J). Our results showed that the presence of b-actin-Neo cassette within GM15884 affects neighboring gene expression and it is therefore essential to excise the cassette prior to obtaining transparent and interpretable subsequent data. Download figure Open in new tab Fig. 4 Expression analysis of Mirc13 neighboring genes. Total RNA was isolated from olfactory bulb (OB) of Mirc13 tm1/tm1 and Mirc13 −/− mice and corresponding negative littermate (Mirc13 +/+ and Mirc13 fl/fl ). Expression of Ptpn6 ( A and F ), Phb2 ( B and G ), Atn1 ( C and H ), Eno2 ( D and I ), and Emg1 ( E and J ) were assessed by RT-qPCR. Data are presented as mean ± SD (*p < 0.05 vs. Mirc13 +/+ control. Light blue column denotes RNA obtained from Mirc13 +/+ ; Yellow column denotes RNA obtained from Mirc13 tm1/tm1 ; dark blue column denotes RNA from Mirc13 fl/fl and red column denotes RNA isolated from Mirc13 −/− mice. Expression Analysis of miR-141-3p and miR-200c-3p in Mirc13 −/− mice We further characterized expression of miR-141-3p and miR-200c-3p in Mirc13 −/− and their corresponding Mirc13 fl/fl mice in the brain. We isolated total RNA from different regions of the brain, including olfactory bulb (OB), frontal cortex (FC), parietal cortex (PC), hippocampus (HP), and cerebellum (CB), from mice of both genotypes. Our results showed both miR-141-3p and miR-200c-3p were highly expressed in the OB (ΔCT 10) ( Fig 5A and B ). Notably, the OB of Mirc13 −/− mice showed no detectable expression of miR-141-3p by RNA-FISH ( Fig 5C ). This finding was further confirmed by RT-qPCR, which demonstrated a marked reduction of both miRNAs (miR-141-3p & miR-200c-3p) in the OB almost negligible ( Fig 5D and I ), along with significantly decreased expression in the FC ( Fig 5E and J ), PC ( Fig 5F and K ), HP ( Fig 5G and L ), and CB ( Fig 5H and M ) of Mirc13 −/− mice as compared to Mirc13 fl/fl mice. Download figure Open in new tab Fig. 5 Expression analysis of miR-141-3p and miR-200c-3p in Mirc13 fl/fl and Mirc13 −/− mice. Total RNA was isolated from olfactory bulb (OB), frontal cortex (FC), parietal cortex (PC), hippocampus (HP), and cerebellum (CB) of Mirc13 fl/fl and Mirc13 −/− mice. Basal expression levels of miR-141-3p ( A ) and miR-200c-3p ( B ) were assessed by RT-qPCR using RNA isolated from Mirc13 fl/fl mice. ( C ) RNA-FISH was performed to detect expression of miR-141-3p in OB of Mirc13 fl/fl and Mirc13 −/− mice. Total RNAs isolated from OB ( D and I ); FC ( E and J ); PC ( F and K ); HP ( G and L ) and CB ( H and M ) were examined for the relative level of miR-141-3p and miR-200c-3p, respectively, by RT-qPCR from Mirc13 fl/fl (dark blue column) and Mirc13 −/− (red column) mice. Data are presented as mean ± SD (*p < 0.05 vs. Mirc13 fl/fl control). DISCUSSION Generation of the Mirc13 tm1 line by Park et al. 2012provided the first genetically engineered model for the miR-141/200c cluster( Park, et al. 2012 ). In their design, a lacZ reporter in association with the En2 splice acceptor and ires sequence, which is similar to the gene trap design( Skarnes, et al. 1992 ), was aimed to reveal spatial expression of the disrupted endogenous GM15884. Expression of the lacZ reporter is under the transcriptional control of the endogenous GM15884 gene, and with the En2 splice acceptor, lacZ sequence will fuse with exon 1 coding sequence of GM15884. Thus, x-gal staining of the lacZ gene product, β-galactosidase, will faithfully recapitulate the spatiotemporal expression of GM15884( Skarnes, et al. 1992 ) as well as the miR-141/200c cluster. GM15884 is a lncRNA gene predicted by bioinformatics as described in the Mouse Genome Database( Eppig, et al. 2005 ) ( https://www.informatics.jax.org/marker/key/326668 ). However, when we performed a blast search of the mouse Expressed Taq Sequence database (dbEST)( Boguski, et al. 1993 ), we were able to identify a mouse cDNA clone, BB612857, of 646 bp. 5’ sequence of BB612857 is identical to predicted exon 1 and 2 of GM15884 the rest of the 3’-downstream sequence identical to predicted GM15884 intron 2 sequence. It is well established that EST are derived from transcribed genes (the transcriptome) and dbEST was used to identify transcribed sequences without requiring a fully sequenced genome( Boguski, et al. 1993 ). Identification of BB612857 confirms the identity of GM15884. This result, together with the tissue-specific lacZ expression, suggests that expression of GM15884/BB612857, as well as Mirc13, is under the control of a tissue-specific Pol II promoter, and our lacZ expression observed in the olfactory bulb and lung in the mice corroborated with the finding of Park et al. Since the Mirc13 tm1 mouse line was original generated by homologous recombination using ES cells, A β-actin-neo selectable marker was included in the original targeting vector and was retained in the Mirc13 tm1 mouse line. It has been reported that the Neo cassette with a strong ubiquitous promoter could influence neighboring gene expression( Pham, et al. 1996 ) and therefore alter phenotype of the mice( Olson, et al. 1996 ; Pan, et al. 2016 ). Park et al explicitly recommended removal of Neo cassette prior to subsequent use of the mouse line. The Neo cassette together with the lacZ sequence were flanked by FRT sites and could be excised easily by breeding the Mirc13 tm1 mice with FLP mice. Additional methods by which one could excise the Frt flanked lacZ/Neo cassette is either by electroporation of Flpo mRNA into one-cell embryos( Hashimoto and Takemoto 2015 ) or infection of one-cell embryos with recombinant adeno-associated viruses expressing Flpo( Nickl, et al. 2024 ), followed by transferring manipulated embryos into pseudopregnant females for subsequent development. Overall, it is important to generate a functional floxed Mirc13 allele prior to using Cre recombinase for either tissue-specific or global deletion of the miR-141/200c cluster in order to avoid interference from the lacZ/neo elements, including unanticipated knockout of endogenous GM15884 and altering the expression of neighboring genes( McBurney, et al. 2002 ; Branda and Dymecki 2004 ). Our neighboring gene expression analyses further confirm the influence of presence of β-actin-neo cassette on expression of many neighboring genes such as ptpn6, phb2 and atn1 in the Mirc13 tm1/tm1 mice. The genes ptpn6, phb2, and atn1 each play critical roles in brain function. Ptpn6 regulates microglial signaling and helps restrain neuroinflammation; its loss can drive excessive immune activation and neuronal injury( He, et al. 2023 ). Phb2 maintains mitochondrial integrity and neuronal survival, so deletion can impair energy metabolism and cognitive functions( Xu, et al. 2014 ). Atn1 acts as a transcriptional co-regulator in neuronal development and disruption can lead to abnormal differentiation, connectivity, or neurodegeneration( Whitton, et al. 1993 ). Thus, downregulation of these genes as shown in Mirc13 tm1/tm1 mice could have significant consequences for neuronal health and brain function. There is no change in expression of these genes in Mirc13 fl/fl mice, suggesting that it is essential to remove the lacZ/β-actin-Neo cassettes to obtain fully functional Mirc13 cKO mice for meaningful and precise analyses of their functional roles. Despite the clear breeding plan recommended by Park et al( Park, et al. 2012 ), several subsequent studies did not adopt the necessary breeding plan. They treated the Mirc13 tm1 mice, which contain lacZ/β-actin-neo cassettes, as Mirc13 cKO animals and bred them directly with Cre deleters ( Guan, et al. 2018 ; Hoefert, et al. 2018 ; Ji, et al. 2018 ; Liu, et al. 2018 ; Wu, et al. 2019 ; Xu, et al. 2025 ). However, Mirc13 tm1 mice contain three colinear loxP sequences in the same allele ( Fig 1A ). It is well established that in the presence of Cre, this would lead to different combinations of paired loxP sequences and result in different excisions of loxP flanked sequences. As shown in Fig 6 , this could generate three different genotypes dependent on the different pairings of loxP sequences: A). excision of the β-actin-Neo cassette alone; B) excision of Mirc13 alone; and C) excision of β-actin-Neo cassette and Mirc13 together. Nevertheless, in all of these cases, both the endogenous GM15884 and Mirc13 will not express and it is still not possible to decipher the precise functional role of miR-141/200c. Other labs have bred Mirc13 tm1 pups with β-actin-Flpe mice prior to breeding them with Cre deleters. However, FLPe is not codon-optimized for expression in mammals and the level of FLP protein expression is compromised and excision of the b-actin-neo cassette may not take place in one-cell embryo leading to mosaic animals( Tran, et al. 2017 ; Mostofa, et al. 2022 ; Tran, et al. 2024 ). We used Rosa26-FLPo mice, which would overcome the issue of low levels of the FLP gene product( Raymond and Soriano 2007 ; Wu, et al. 2009 ; Kranz, et al. 2010 ). Our breeding plan, as shown in Fig 1 , is to remove the lacZ/neo cassettes using ROSA26-FLPo mice, generating a functional Mirc13 floxed allele prior to using Cre mice, such as the germline Hprt CAG-Cre mice, to achieve global deletion of the Mirc13 allele. These steps ensure that any observed phenotype will be attributed directly to loss of the miR-141/200c cluster, without any confounding contributions from transcriptional silencing or residual neomycin cassette effects. Download figure Open in new tab Fig. 6 Breeding Mirc13 tm1 mice containing three colinear loxP sites with Cre mice could lead to three different genotypes. A Excision of the β-actin-Neo cassette; B excision of β-actin-Neo cassette together with Mirc13 sequence; and C deletion of Mirc13 sequence. In all three different genotypes, both GM15884 and Mirc13 are not expressed due to the presence of the lacZ polyA sequence. This breeding plan is not merely technical but critical for precise evaluation of the miR-141/200c functional roles. Studies of the miR-200 family—including their roles in late postnatal neurogenesis( Beclin, et al. 2016 ), stem cell differentiation( Zhang, et al. 2015 ), and cancer biology( Chen, et al. 2013 ; Jin, et al. 2017 ; Tseng, et al. 2017 ) rely strongly on precise gene dosage and context. Using animals with imprecise genotypes will only hamper validities of the data leading to spurious conclusion and discrepancy among different reports. By adhering to the breeding plan according to the design of the original targeting vector, our work provides a framework to generate unambiguous global and functional conditional knockouts of the miR-141/200c cluster. Author contributions RV conceptualized the project. SKY, RS, RV and SPY wrote the manuscript. MKC, RS, SKY and KL performed the experiments. RV and SPY supervised the work. RV secured the funding for the project. All the authors approved the work. Ethics declarations Ethical Approval All experiments were approved and conducted in compliance with the protocols approved (Number: AP-201043-0926) by the Institutional Animal Care and Use Committee (IACUC) at UConn Health. Animal data was reported following the ARRIVE Guidelines. Declaration of Conflicting Interests The authors declared no conflicts of interest. Funding This work was supported by NIH, United States (1R21NS114981 and R01 NS140176) and REP Convergence (OVPR UConn, United States) grant to R.V. Data Availability The data supporting the findings of this study will be available on request from the corresponding author. Acknowledgments ACKOWLEDGEMENT Funder Information Declared NIH Common Fund , 1R21NS114981 , R01 NS140176 Footnotes Data is more clear and easy to present additional summary figure (Fig 6) is added REFERENCES 1. ↵ Beclin C , Follert P , Stappers E , Barral S , Coré N , Chevigny A , Magnone V , Lebrigand K , Bissels U , Huylebroeck D , et al. 2016 . 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Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele Sanjeev Kumar Yadav , Rashmi Srivastava , Mary-Katherine Cormier , Katie Lowther , Siu-Pok Yee , Rajkumar Verma bioRxiv 2025.09.02.673774; doi: https://doi.org/10.1101/2025.09.02.673774 Share This Article: Copy Citation Tools Generation of miR-141/200c conditional knockout mice from knockout-first, reporter-tagged parent and functional validation of the floxed allele Sanjeev Kumar Yadav , Rashmi Srivastava , Mary-Katherine Cormier , Katie Lowther , Siu-Pok Yee , Rajkumar Verma bioRxiv 2025.09.02.673774; doi: https://doi.org/10.1101/2025.09.02.673774 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)