Functional analysis of a role of prolylendopeptidase in brain by constructing gene targeting mice

小鼠脯氨酸多肽酶由肽酶结构域（n端和c端）和螺旋桨结构域（中间区域）组成。编码该蛋白的基因由15个外显子组成。其中外显子6编码螺旋桨结构域。我们试图构建缺乏脯氨酸多肽酶基因外显子6的突变小鼠。首先，我们从129个品系小鼠的基因组文库中克隆了该基因。接下来，我们构建了一个靶向载体，其外显子6被新霉素耐药基因的插入破坏。通过电穿孔将靶向载体导入小鼠胚胎干细胞，然后通过同源重组破坏内多肽酶基因。突变的胚胎干细胞用于构建嵌合体小鼠。我们现在正通过对Fl小鼠的分析来测试突变ES细胞的种系传播。我们还测试了胚胎干细胞是否缺乏脯氨酸多肽酶基因的两个等位基因。结果表明，我们可以构建一个不表达脯氨酸多肽酶活性的零突变体。突变体的生长与野生型胚胎干细胞的生长难以区分，表明该蛋白对胚胎干细胞的增殖是必不可少的。

Mouse prolylendopeptidase consists of the peptidase domain (N-terminal and C-terminal) and the propeller domain (intermediate region). The gene encoding this protein consists of 15 exons. Among them, the exon 6 encodes the propeller domain. We tried to construct mutant mice lacking the exon 6 of the prolylendopeptidase gene. First, we cloned the gene from the genomic library of 129 strain mice. We next constructed a targeting vector whose exon 6 was disrupted by the insertion of the neomycine-resistant gene. The targeting vector was introduced into mice ES cells by electroporation, followed by disruption of the endopeptidase gene by homologous recombination. The mutant ES cells were used for construction of chimera mice. We are now testing the germ line transmission of the mutant ES cells by analysis of Fl mice. We also tested whether ES cells lacking both alleles of the prolylendopeptidase gene. The results showed that we could construct a null mutant which expressed no prolylendopeptidase activity. The growth of the mutant was indistinguishable from that of wild type ES cells, indicating that the protein is dispensable for proliferation of ES cells.