Deletion of kallikrein 1b5 ( Klk1b5 ) has no impact on fertility in mice

Kallikreins (KLKs) are a family of serine proteases responsible for many physiological functions in mammals. Of the various family members, KLK1 is well studied and found to be highly conserved among mammalian species. Aberrant gene expression of proteins in the KLK family was linked to asthma, hyperkalemia, artery dysfunction, and many other diseases (Prassas, Eissa, Poda, & Diamandis, 2015). KLK2‐15 are known as kallikrein‐related peptidases, which are expressed in a wide range of tissues with specialized roles. In rodents, an evolutionary gene duplication resulted in insertion of 13 additional gene‐encoding KLK1‐related peptidase subfamily members (Klk1b1‐27) between Klk1 and Klk15 loci (Olsson & Lundwall, 2002). In the uterus, the expression of Klk1 and its subfamily members are estrogen (E2)‐dependent. Accordingly, our previous work showed that Klk1b5 is expressed at the highest level compared to other Klk members in the mouse uterus after E2 treatment (Li, Garcia, Gewiss, & Winuthayanon, 2017). However, the physiological function of Klk1b5 has never been investigated. As such, we generated a loss‐of‐function mouse model using CRISPR/Cas9 technology to determine the biological function of Klk1b5. A single‐guide RNA was generated to target the beginning of the mature peptide coding sequence of exon 2 of the Klk1b5 gene (Figure 1a). As a result, a 19 base pairs (bp) deletion was introduced to the 5′ end of exon 2, causing a frameshift mutation (Figure 1a, bottom sequences). Genotyping of Klk1b5 alleles showed the amplicon with the product size of 290 bp, Klk1b5 with 271 bp, and Klk1b5 with both 290 and 271 bp (Figure. 1b). Because the antibodies required to differentiate KLK1B5 from other KLK1Bs are not available, quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) was used to validate the deletion of Klk1b5 and the expression of other Klk1b family genes in highly expressed tissues such as the submandibular gland. As anticipated, Klk1b5 mRNA was not detectable in the submandibular glands of Klk1b5 mice (Figure 1c). Klk1 was significantly reduced in Klk1b5 and Klk1b5 compared to Klk1b5 mice. Conversely, expression of Klk1b24 was significantly elevated in Klk1b5 and Klk1b5 tissues. There were also no significant differences in body weight between Klk1b5, Klk1b5, and Klk1b5 male or female mice (Figure 1d). In addition, an evaluation of the histoarchitecture of the submandibular gland and the uterus revealed no morphological differences between Klk1b5 and Klk1b5 mice (Figure 1e). The fecundity of Klk1b5 and Klk1b5 mice was also determined using a breeding trial that occurred over the course of 6 consecutive months. No fertility defects in Klk1b5 male or female mice compared to the Klk1b5 or Klk1b5 controls were observed (Figure 1f). Based on these findings, we conclude that the loss of Klk1b5 did not disrupt male or female fertility. However, the results of our quantitative RT‐PCR suggest that this lack of a fertility phenotype in the Klk1b5 mice may be due to compensation by Klk1b24. Compensation by other Klk subfamily members could also have rescued the other physiological abnormalities that may have been present in the Klk1b5 mice. To test this, we are currently generating a mouse model with a deletion of both Klk1b5 and Klk1b24 genes to determine if an increase in Klk1b24 level compensates for the uterine function of Klk1b5. If this is the case, we would observe a fertility defect in Klk1b5; Klk1b24 female mice.