反义阻断hMMS2基因表达以抑制细胞生长

Antisense inhibition of hMMS2 gene expression suppresses cell growth

将新近发现的人泛素缀合酶样蛋白基因h MMS2的 c DNA特定反向克隆到本室改建的真核细胞表达载体 p MAMneo- amp-中 ,构建了表达h MMS2反义 RNA的重组质粒 .将此反义表达重组质粒 (p MAM- anti- h MMS2 )用改良的磷酸钙法转染人羊膜 FL细胞 ,建立 FL- h MMS2 -细胞系 .比较该细胞系及 FL细胞和转染了空载体 p MAMneo-amp-的 FL细胞 (FL- MAMneo细胞 )的生长速度 ,发现 FL- h MMS2 -细胞系在地塞米松诱导下h MMS2基因表达被反义抑制后导致细胞生长受阻 ,提示 h MMS2基因为细胞生长所必需 .

The appropriate length cDNA fragment of the recently identified human gene hMMS2 which encodes ubiquitin- conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp -. The recombinant plasmid which can express hMMS2 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-anti-hMMS2 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and the G418 resistant FL-hMMS2 - cell line was established. It was found that the growth rate of the FL-hMMS2 - cell line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp -. The result of cell growth suppression by antisense inhibition of hMMS2 gene expression suggests that hMMS2 gene be an essential gene for cell growth.