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

利用多组寡引物，对生长抑素受体SSTR2和SSTR5基因组DNA片段进行了亚克隆。获得3.8Kb的SSTR2片段和4.2Kb的sstr5片段。两个片段都包含一小部分5′序列和受体编码区。将Neo-cassette插入编码区，用HSV-tK将片段亚克隆到pMCtk载体上。将这些载体转染到胚胎干细胞中，先用Neo-resistance筛选克隆，然后用无环鸟苷进行阳性选择。在这一步之后，选择35个克隆，用同源重组位点以外的引物进行PCR， 35个克隆中有3个克隆与转染的DNA片段成功重组（2个克隆为SSTR2, 1个克隆为SSTR5）。将这3个克隆注射到小鼠胚胎中。从皮毛斑块来看，我们得到了杂合的F0，但在F1代中，我们没有得到纯合的敲除小鼠。

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.