Deletion of kallikrein 1b5 (Klk1b5) has no impact on fertility in mice.
Deletion of kallikrein 1b5 (Klk1b5) has no impact on fertility in mice.
复制标题
删除激肽释放酶 1b5 (Klk1b5) 对小鼠的生育能力没有影响。
DOI：
10.1002/mrd.23145
复制
发表时间：
2019
影响因子：
2.5
通讯作者：
Winuthayanon,Wipawee
中科院分区：
文献类型：
--
作者：
Li,Shuai;Beedle,My-Thanh;Herrera,GerardoB;Tam,KeilaK;Winuthayanon,Wipawee
激肽释放酶（KLK）是哺乳动物中负责许多生理功能的丝氨酸蛋白酶家族。在各种家族成员中，KLK 1被充分研究，并发现在哺乳动物物种中高度保守。KLK家族中蛋白质的异常基因表达与哮喘、高钾血症、动脉功能障碍和许多其他疾病有关（Prassas，Eissa，Poda和Diamandis，2015）。KLK 2 - 15被称为激肽释放酶相关肽酶，其在广泛的组织中表达，具有特定的作用。在啮齿类动物中，进化基因重复导致在Klk1和Klk15基因座之间插入13个额外的编码Klk1相关肽酶亚家族成员（Klk1b1 - 27）的基因（Olsson & Lundwall，2002）。因此，我们以前的工作表明，与E2治疗后小鼠子宫中的其他Klk成员相比，Klk1b5的表达水平最高（Li，Garcia，Gewiss，& Winuthayanon，2017）。然而，Klk1b5的生理功能从未被研究过。因此，我们使用CRISPR/Cas9技术生成了功能丧失小鼠模型，以确定Klk1b5的生物学功能。生成单向导RNA（sgRNA），以靶向Klk1b5基因外显子2的成熟肽编码序列的开始（图1a）。结果，在外显子2的5 '端引入19 bp缺失，导致移码突变（图1a，底部序列）。Klk1b5 +/+等位基因的基因分型显示扩增子的产物大小为290 bp，Klk1b5 −/−为271 bp，Klk1b5 +/−为290和271 bp（图）。1 b）。
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 were linked to asthma, hyperkalemia, artery dysfunction, and many other diseases (Prassas, Eissa, Poda, & Diamandis, 2015). KLK2–15 are known as kallikreinrelated peptidases, which are expressed in a wide range of tissues with specialized roles. In rodents, an evolutionary gene duplication resulted in an insertion of 13 additional geneencoding 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 (sgRNA) 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 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).