ESTABLISHMENT AND ANALYSIS OF SOMATOSTATIN RECEPTORS (SSTR2 AND SSTR5) KNOCKOUT MOUSE

我们利用PCR技术，用几组寡聚引物亚克隆了生长抑素受体SSTR 2和SSTR 5基因组DNA片段。获得了3.8Kb的SSTR 2片段和4.2Kb的SSTR 5片段。这两个片段都含有小部分5 '端的侧翼序列和受体编码区。将Neo-cassette插入到编码区，亚克隆到含有HSV-tK的pMCtk载体中，转染ES细胞，经Neo-resistance筛选后，再用阿昔洛韦进行阳性筛选。在此步骤之后，选择35个克隆，并且用同源重组位点外的引物进行PCR，显示35个克隆中的3个成功地与转染的DNA片段重组（两个克隆用于SSTR 2，一个用于SSTR 5）。将这3个克隆注射到小鼠胚胎中。F0代小鼠的毛斑为杂合子，F1代小鼠的毛斑为杂合子，F1代小鼠的毛斑为杂合子。

We subcloned genomic DNA fragment of somatostatin receptor, SSTR2 and SSTR5, with PCR using several sets of oligoprimers. 3.8Kb Fragment of SSTR2 and 4.2Kb fragment of SSTR 5 were obtained. Both fragment consist with small portion of 5'franking sequence and receptor coding regions. Neo-cassette was inserted into the coding region and the fragments were subcloned into the pMCtk vector with HSV-tK.Those vectors were transfected to the ES cells, then, clones were selected by Neo-resistance first followed by positive selection with acyclovir. After this step, 35 clones were selected and PCR with primers outside the homologously recombined site revealed that 3 out of 35 clones were successfully recombinated with trasfected DNA Fragment (two clones for SSTR2 and one for SSTR5). These 3 clones were injected to mouse embryo. We got heterozygous F0 judging from the fur patches, however, failed to get homozygous knockout mice in F1 generations.