Male Keratin 13‐Cre Expression Causes Unanticipated Germline Deletion in <i>Notch1</i> Floxed Mice

Notch1 receptor activation plays an important role in epithelial homeostasis and immunoregulation; however, its role in oral mucosal immunity remains unknown. Testing the impact of epithelial Notch1 activation in oral innate responses, including antimicrobial proteins like phospholipase A₂ group IIA (PLA₂-IIA), requires epithelial-specific Notch1 knockout mice. Creating Balb/c; K13Cre; Notch1^Flox/Flox mice was initiated by crossing C57BL/6; K13Cre; Notch1^Flox/Flox mice (naturally deficient for PLA₂-IIA), expressing Cre recombinase under the control of the Krt13 (keratin 13) promoter (K13Cre) and Notch1^Flox/Flox alleles to PLA₂-IIA expressing Balb/c mice to generate Balb/c:C57BL/6; K13Cre; Notch1^Flox heterozygotes and to ultimately create Balb/c; K13Cre; Notch1^Flox/Flox (Notch1 deficient) and Balb/c; Notch1^Flox/Flox WT littermate controls. While all first-generation non-K13Cre expressing mice had the expected Notch1^Flox heterozygote Notch1 genotype, surprisingly, all first-generation K13Cre expressing mice had an apparent K13Cre; Notch1 homozygote genotype, which is impossible when crossing Notch1 and Notch1^Flox/Flox homozygotes. We found that the absence of the floxed Notch1 PCR amplicon resulted from recombination between the two LoxP sites flanking the forward Notch1 PCR primer, and the recombined Notch1 allele was present in the germline of first-generation congenic mice. Finally, we show that this unexpected recombination is avoided by using female K13Cre; Notch1^Flox donors and is a cautionary reminder that individual promoters are expressed during spermiogenesis.