| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 90 === We have isolated a mouse gene msbk , which is homologous to zebrafish bsk146 gene and rat sbk gene , from mouse brain cDNA . It encodes a 417 amino acid residues as a serine/threonine protein kinase . It’s C-terminal end contains a proline-rich region , which included a SH3 bind core and a nuclear localization signal ( NLS ) . We have constructed msbk-FL ( wild type msbk ) , msbk-KR ( ATP binding site mutation form ) , msbk-D ( C-terminal end deletion form ) and cloned in HA-yun vector . Then we transfected these plasmids into COS1、C6、NG108-15 and HepG2 cell lines . By immunostaining , we observed that mSBK-FL only expressed in cytoplasm of NG108-15 and HepG2 cells . But in COS1 and C6 cells , mSBK-FL expressed both in cytoplasm and nucleus . And mSBK-KR and mSBK-D only expressed in cytoplasm of these cell lines. It may reveal that mskb expressed differently in distinct cell lines . And we can guess that phosphorylation and C-terminal end , contained a NLS and SH3 binding core , may affect the cellular localization of mSBK . In order to analyze the function of msbk in vivo , we also cloned a mouse genomic fragment about 15kb including msbk gene . We replaced the msbk exon1 with GK-Neo . Then we cloned this genomic fragment into a pBSK-sk+ vector to be a targeting vector for msbk knock-out mice . Finally we have cloned a msbk 5’flanking sequence about 5.5 kb . By CAT-basic vector , we performed promoter assay and found that the msbk promoter was located at -613 ~ +240 . Of these three kinds of cell lines that we used , the msbk promoter activity in NG108 cells was the strongest .
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