FUNCTIONAL ANALYSIS OF CA/CALMODULIN-DEPENDENT PROTEIN KINASE II IN PANCREATIC BETS-CELLS WITH CONDITIONAL KNOCKOUT MICE.

我们已经报道了钙/钙调素依赖性蛋白激酶II（CaM激酶II）参与胰岛素分泌。本研究的目的是进一步分析CaM激酶II在胰腺β细胞中的作用。从C57/BL 6小鼠基因组文库中克隆了CaM激酶Ⅱ δ亚基基因组，经鉴定含有编码起始密码子的外显子。分析限制性内切酶位点，在外显子的上游（Nae I）或下游（Sex AI，Pfl MI）鉴定出三个独特的限制性内切酶位点。扩增loxP片段，在两端连接Nae I识别序列，并插入克隆的Nae I位点。进一步将连接有lox P和Sex AI序列的胸苷激酶cDNA和白喉毒素B cDNA插入该克隆构建靶向载体。此外，从MIN 6细胞中分离出CaM激酶II（CaM KIIN）cDNA的内源性抑制肽。制备CaM KIIN的腺病毒表达载体，分析CaM激酶II在β细胞中的作用。此外，用荧光素酶报告基因分析了CaM激酶II δ亚基基因的启动子区。TATA样序列上游2.1kb的区域似乎含有启动子活性。在该区域确定了可能的转录因子结合位点。

We have reported that Ca/calmodulin-dependent protein kinase II(CaM kinase II) was involved in insulin secretion. The aim of this study is to further analyze the roles of CaM kinase II in pancreatic beta cells. The 19 kb genomic clone of CaM kinase II delta subunit was isolated from C57/BL6 mouse genomic library and ascertained to contain the exon that encoded initial codon. Restriction enzyme sites were analyzed and three unique restriction enzyme sites were identified at upsteam(Nae I) or downstream(Sex AI, Pfl MI) of the exon. A lox P fragment was amplified, attached by Nae I recognition sequences in both ends and inserted into Nae I site of the clone. Furthermore, thymidine kinase cDNA attached by lox P and Sex AI sequence and diphtheria toxin B cDNA were planned to insert the clone to construct targeting vector. Also endogenous inhibitory peptide for CaM kinase II(CaM KIIN) cDNA was isolated from MIN6 cells. Adenoviral expression vector of CaM KIIN was preparing to analyze the role of CaM kinase II in beta cells. Moreover, promoter region of the CaM kinase II delta subunit gene was analyzed with luciferase reporter gene. The 2.1 kb upstream region from TATA-like sequence seemed to contain promoter activity. Possible transcriptional factor binding sites were identified in the region.