A Cxcl15 Cre Recombinase Mouse Model Useful to Study Gland Development in the Uterus.

All experimental procedures were approved by the University of Missouri’s Institutional Animal Care and Use Committee and were performed according to the guidelines set forth in the Guide for the Use and Care of Laboratory Animals. C57BL/6J embryo donor females (3 weeks of age) and stud males (10 weeks of age) were purchased from Jackson Laboratory. CD-1 surrogate females (8 weeks of age) were purchased from Charles River. Foxa2 (stock number: 022620) and Rosa26nTnG (stock number: 021309) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were kept on a 12:12 light cycle. Food and water were available ad libitum . Control ( Cxcl15 +/+ ;Foxa2 f/f ) and Cxcl15 Cre/+ ;Foxa2 f/f 8-week-old female mice were housed individually and continuously with proven-fertile CD-1 male mice. Mating was confirmed by presence of a vaginal plug. Fertility was assessed by monitoring litter frequency and size for 6 months. The C57BL/6 mouse genomic sequence for chemokine (C-X-C motif) ligand 15 (Cxcl15) was obtained from Ensembl.org (assembly GRCm39). The Cas-OFFinder tool, maintained by the Center for Genome Engineering Institute, was used to calculate off target scores and design sgRNAs ( Bae et al., 2014 ). A sgRNA targeting the ATG start codon located in Exon 1 of Cxcl15 was designed using the CCTop website maintained by the Centre for Organismal Studies ( Stemmer et al., 2015 ) that calculates CRISPRater efficiency prediction scores for the sgRNA ( Labuhn et al., 2018 ). The sgRNA (5’-TGAGCAGCCATGTCCGTCTG-3’) was ordered as a chemically modified synthetic sgRNA through Synthego with 2′-O-methyl analogs and 3′ phosphorothioate internucleotide linkages at the first three 5′ and 3′ terminal RNA residues. For CRISPR-Cas9 mediated knock-in, a repair template was designed to insert the iCre-pA cassette into the endogenous Cxcl15 locus. The repair template was synthesized by VectorBuilder with asymmetrical homology arms (627 bp and 700 bp, respectively) and packaged into an ssAAV1 vector. Following a published protocol ( Davis et al., 2023 ), C57BL/6J zygotes with visible pronuclei were placed in 30 μL KSOM medium containing 3×10 7 GC/μL ssAAV1-Cxcl15-iCre under mineral oil in 35 mm Petri dish and cultured at 37°C with 5% CO 2 and maximal humidity for 18 to 20 h. The following day two-cell stage embryos were electroporated with CRISPR sgRNA/Cas9 RNP complexes. RNP complexes were formed by mixing 100 ng/μL sgRNA +100 ng/μL enhanced-specificity Cas9 protein with Opti-MEM ™ as buffer in the reagent mix and incubated at room temperature for 10 min immediately prior to electroporation. Using a NepaGene21 electroporator with a 1.5-mm gap glass slide electrode, two-cell embryos were electroporated using the following conditions: Poring pulse: 40 V, 3.5 ms length, 50 ms interval, 10% decay rate, positive polarity (x4 pulses); and Transfer pulse: 5 V, 50 ms length, 50 ms interval, 40% decay rate, alternating polarity (x5 pulses). Electroporated embryos were surgically transferred to pseudopregnant surrogate females the same day as the electroporation. Uteri were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 40 min at 4°C, and cryoprotected by overnight immersion in 15% sucrose/PBS) at 4°C. Tissue was embedded in optimal cutting temperature compound (OCT) and cryo-sectioned at 7 μm. Uteri from Cxcl15 Cre/+ Foxa2 f/f and control mice were fixed in 4% paraformaldehyde in PBS overnight at room temperature, embedded in paraffin, and sectioned at 5 μm. The OCT and paraffin-embedded tissue sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA. Cat No: 22037246) and stored at −20°C and room temperature, respectively. Immunofluorescent analysis of paraffin-embedded sections was conducted using previously described methods ( Rizo et al., 2023 ). Briefly, slides were dried at 60°C for 20 min, deparaffinized in xylenes, and rehydrated through an ethanol gradient. Next, slides were boiled in 0.1 M Sodium Citrate antigen retrieval buffer (pH 6.0). Subsequently, slides were washed in PBS, blocked in 2.5% normal goat serum (NGS), and incubated with primary antibodies to Cytokeratin 8 (1:200 dilution; University of Iowa Developmental Studies Hybridoma Bank, TROMA-1;), CXCL15 (1:500 dilution; Abcam, Cat No. ab197016) and FOXA2 (1:700 dilution; Abcam, Cat No. ab108422) overnight at 4°C. Following primary antibody incubation, slides were thoroughly washed with PBS and incubated with Alexa Fluor conjugated secondary goat anti-rabbit and anti-rat antibodies (1:500 dilution) for 1 h at room temperature. Secondary antibodies were removed by a series of washes in PBS, nuclei were stained with Hoechst 33342 (2 μg/ml; Life Technologies, Carlsbad, CA. Cat No. H3570) for 5 min, and cover slips affixed with Diamond Mount (Invitrogen, P36961 ). Cryosections were dried at 45°C for 30 min, permeabilized with 0.1% Triton X-100/PBS for 5 min, blocked in 2% NGS, and incubated with primary antibodies to cytokeratin 8 (TROMA-1; 1:200 dilution, University of Iowa Developmental Studies Hybridoma Bank) and FOXA2 (1:700 dilution Abcam, Cat No: ab108422) overnight at 4°C. Slides were washed in PBS and incubated with Alexa Fluor conjugated goat anti-rabbit and anti-rat secondary antibodies. Next, excess of unconjugated Fab antibody raised against rabbit was used to mask the rabbit IgG (Jackson ImmunoResearch, Cat No: 111–007–003). Slides were washed in PBS and incubated with a primary antibody to GFP (1:100 dilution Invitrogen, Cat No: G10362 ) in 2%NGS/PBS for 30 min at RT, washed in PBS, and incubated with Alexa Flour goat anti-rabbit secondary for 30 min at room temperature. Nuclei were stained and cover slipped as described above. All images were acquired with a Leica DM5500 B upright microscope using the Leica Application Suite X (LAS X).

Cxcl15 expression in the developing uterus was determined using single-cell RNA-seq datasets from postnatal days (PND) 1, 5, 12, and 15 ( Spencer et al., 2023 ). Cxcl15 expression overlapped prominently with the well-established GE marker Foxa2 ( Kelleher et al., 2017 ) in UMAP plots, consistent with gland formation during postnatal uterine development ( Figure 1A ). Further, Cxcl15 was identified as a top differentially expressed gene in an existing RNA-seq study comparing mice with (Control), without uterine glands ( Pgr Cre/+ Foxa2 f/f ), or adult Foxa2 conditional knockout ( Ltf Cre/+ Foxa2 f/f ) on GD 4 ( Figure 1B ) ( Kelleher et al., 2018 ). Cxcl15 protein was localized specifically to the nascent GE cells in the PND 5, 12, 15, and 20 neonatal uterus as well as mature GE cells in the GD 4 adult uterus ( Figure 1C ). In addition, Cxcl15 was observed in the epithelium of the vagina and cervix, but not in the oviduct or ovary, from GD 4 mice ( Figure 1D ). These results suggest that Cxcl15 is a preferred candidate for generating a GE-specific Cre driver mouse to facilitate studies of uterine gland biology. The Cxcl15 gene contains four exons, and loss of both alleles does not affect viability or fertility of mice ( Chen et al., 2001 ). The approach was to insert the iCre recombinase coding sequence into the endogenous ATG start site of the mouse Cxcl15 gene (ENSMUSG00000029375.7) using CRISPR-mediated homology-directed repair ( Figure 2A ). Genome editing was accomplished by injecting CRISPR reagents directly into zygotes to generate founder animals. Correctly targeted founders were subsequently backcrossed to C57BL/6 mates for germline transmission, producing heterozygous offspring bearing the Cxcl15 - iCre allele. Heterozygous mice were then intercrossed to generate animals homozygous for the Cxcl15-iCre allele. Breeding male and female Cxcl15 Cre/+ mice produced all three possible genotypes ( Figure 2B ) with normal litter sizes. As expected, normal fertility was observed when homozygous Cxcl15 Cre/Cre males and females were mated together ( Figure 2C ). Thus, homozygous and heterozygous Cxcl15-iCre female and male mice are viable and fertile. To define the temporal onset and cell specificity of Cxcl15-iCre activity, Cxcl15 Cre/+ mice were crossed with the Rosa26 nT/nG reporter line (strain number 023035) in which nuclear GFP is expressed upon Cre-mediated excision of a floxed stop cassette. Epifluorescence analysis of the neonatal mouse uterus found no Cre activity at PND 5, whereas GFP-positive GE cells emerged and increased between PNDs 12 and 15 ( Figure 3A ). All GE cells were GFP-positive in the PND 20 and adult GD 4 uterus. Importantly, no GFP reporter activity was observed in the LE, stroma, or myometrium of the neonatal as well as adult uterus ( Figure 3A ). In adult GD 4 mice, Cre activity was observed in the epithelium of the vagina/cervix but not in the oviduct or ovary ( Figure 3B ). The functional utility of this new model was then assessed by crossing Cxcl15 Cre/+ mice with Foxa2 f/f mice. In the neonatal mouse uterus, FOXA2 is expressed exclusively in GE cells as they differentiate and bifurcate from the LE ( Kelleher et al., 2019 ). Conditional deletion of Foxa2 from the uterine epithelium during the first week of life using the Pgr -Cre model completely inhibited GE differentiation ( Jeong et al., 2010 ; Kelleher et al., 2017 ). In Cxcl15 Cre/+ mice, Cre activity in GE begins after the cells differentiate ( Figure 3A ). Immunofluorescence analyses of Foxa2 in the Cxcl15-iCre ; Foxa2 conditional knockout uterus revealed a loss of Foxa2 from some GE cells by PND 15 ( Figure 4 ). In contrast, Foxa2 was absent from the GE of the adult GD 4 uterus ( Figure 4 ). The adult Cxcl15-iCre ; Foxa2 f/f conditional knockout mice were infertile, with none of the 5 females producing any litters over a six-month breeding trial (data not shown). Of note, adult mice lacking either glands or Foxa2 in their glands are infertile due to the lack of leukemia inhibitory factor (Lif) expression by the GE during early pregnancy ( Jia et al., 2025 ; Kelleher et al., 2018 ; Kelleher et al., 2017 ). In fact, Lif supplementation rescues embryo implantation and pregnancy in Foxa2-deficient gland-containing mice. These results are consistent with previous findings that Foxa2 is required for uterine gland genesis but not maintenance of the GE ( Kelleher et al., 2017 ). Taken together, these data establish Cxcl15 - iCre as a new tool for targeted gene manipulation in developing and adult uterine glands. The ability to delete or overexpress candidate genes at specific developmental intervals using Cxcl15 -iCre is anticipated to substantially enhance mechanistic insights into uterine gland biology.

The uterus is an epitheliomesenchymal organ with three major tissue compartments (epithelium, stroma and myometrium) and two major epithelial cell types (luminal epithelium or LE and glandular epithelium or GE) in the endometrium ( Kelleher et al., 2019 ). The glands of the endometrium develop only or primarily after birth. In mice and domestic animals, uterine glands have established biological roles in regulating implantation and pregnancy establishment ( Kelleher et al., 2019 ; Spencer et al., 2019 ). In humans, uterine glands are also considered essential for pregnancy and are also involved in the most prevalent type of uterine cancer (endometrial adenocarcinoma), and other diseases such as adenomyosis and endometriosis, that negatively affect the fertility and health of women ( Chen et al., 2022 ; Parasar et al., 2017 ; Santulli et al., 2025 ; Urick & Bell, 2019 ; Wang et al., 2020 ). Beyond their roles in pregnancy, the glands have biological roles in endometrial regeneration during the menstrual cycle and after parturition ( Cousins, Filby, et al., 2021 ; Cousins, Pandoy, et al., 2021 ). Uterine gland morphogenesis is primarily or uniquely a postnatal event ( Kelleher et al., 2019 ; Spencer et al., 2012 ; Vue et al., 2018 ). The newborn mouse uterus lacks glands and consists of a simple unspecified epithelium supported by undifferentiated mesenchyme. Between postnatal days (PND) 6 and 9, the epithelium becomes specified into uterine type LE and GE cells begin to differentiate and bifurcate. By PND 12, gland buds extend from the LE into the surrounding endometrial stroma as they develop toward the myometrium. The glands are well developed in the uterus by PND 20. Despite their fundamental role in reproduction and tissue homeostasis, the cellular and molecular mechanisms governing uterine gland development, morphogenesis, and function remain incomplete. Dissecting the function of GE specific and expressed genes and their products in vivo remains challenging, partially due to limitations in the availability of animal models that provide precise temporal and spatial control over gene expression in this cell population. Several mouse models have been developed that express Cre recombinase in uterine epithelia, but each has limitations for gland-specific studies. Pgr-Cre mice exhibit Cre activity in all major uterine compartments (epithelium, stroma, myometrium) and other reproductive tract organs ( Contreras et al., 2010 ; Daikoku et al., 2014 ; Soyal et al., 2005 ; Uetzmann et al., 2008 ; Winuthayanon et al., 2010 ). Ltf-iCre and Sprr2f-Cre mice display non-uniform recombination in the epithelia of the adult uterus and, like Wnt7a-Cre mice, are expressed in all uterine epithelium with ectopic activity in multiple organs ( Contreras et al., 2010 ; Daikoku et al., 2014 ). While Foxa2-iCre mice may have GE-specific activity in neonatal and adult uterus, their utility is limited by widespread expression in embryonic endoderm and endoderm-derived organs including lung, liver, pancreas, and gastrointestinal tract ( Uetzmann et al., 2008 ). Thus, there remains a critical need for GE-specific Cre driver mouse line.. To date, only a small number of genes are known to be expressed exclusively in the GE of the developing and adult mouse uterus ( Filant & Spencer, 2013 ; Kelleher et al., 2019 ; Tang & Rancourt, 2005 ). Among those, C-X-C motif chemokine ligand 15 ( Cxcl15 ) is an attractive candidate for generating a GE-specific Cre driver line. The Cxcl15 gene is expressed specifically in the GE of the developing and adult mouse uterus ( Filant & Spencer, 2013 ; Jia et al., 2025 ; Schmitz et al., 2007 ). Importantly, mice with a global deletion of Cxcl15 exhibit normal viability and fertility ( Chen et al., 2001 ). The spatiotemporal expression pattern of Cxcl15 in the mouse uterus, combined with its non-essential role in normal physiology, makes it an ideal target to generate a uterine GE-specific Cre mouse line conditional ablation or overexpression of genes specifically in the glands of the developing neonatal uterus. Utilizing CRISPR/Cas9-based genome editing, improved Cre recombinase (iCre) was inserted into the Cxcl15 locus. Validation studies using reporter mice and functional studies with conditional knockout of Foxa2 established that Cxcl15-iCre mice are useful for fundamental studies of endometrial gland development and function in the uterus.