【摘要】 本文选取人elp3基因三个不同的区段(基因全序列、5′端非HAT区、3′端HAT区)构建了反义质粒pRS313a1644、pRS314a1107、pRS314a450,通过与人elp3基因共转化酵母后的功能互补和基因表达检测,筛选出可显著抑制人Elp3表达的5′端非HAT区1107bp有效反义片段。该片段在随后的HeLa细胞转染实验中被检测出可显著抑制Elp3的mRNA水平,表明利用该方法可避开繁琐的细胞培养和转染步骤,快速筛选到人类兴趣基因的有效反义片段。更多还原

【Abstract】 Antisense RNAs have often been used to study functions of human genes.In order to obtain the effective antisense fragments,researchers always have to face repeated works including cell culture and transfection etc.Here we chose yeast,Saccharomyces cerevisiae,as a model organism to explore the simple and effective way to obtain antisense fragments targeting human genes.This single-cell eucaryote divides rapidly,can be cultured easily just as E.coli,and possesses many biological processes similar to mammalian.Thus,it is the best choice for the study focus on human gene transcription and other biological processes. Human Elongator complex was found to be very similar to the yeast Elongator both in composition and its interaction with RNA polymerase II.The elp3 subunit of human Elongator can significantly complement the growth defects of Yeast elp3 Delta Strain.So we used yeast complementation systems to select the antisense fragment targeting help3. Three different fragments including full length(a1 644),HAT-deletion(a1 107)and HAT(a450)sequence of help3 were amplified and inserted into pRS313 or pRS314 to construct antisense plasmids pRS313a1 644,pRS314a1 107 and pRS314a450.Through the sensitive assay and RT-PCR detection of help3 and ssa3 expression in yeast,we approved that the HAT-deletion fragment(a1 107)repressed the expression of cotransformed help3 significantly and reduced the complementation ability of help3 dramatically in yeast.Then the a1 107 antisense mammalian expressing plasmid was constructed and transfected into Hela cells.Northern blot assay indicated that a1 107 significantly reduced the expression of help3 in HeLa cells.So we regard that cotransforming targeting human gene and its antisense fragments into yeast and obtaining the significant reduction expression of targeting human gene is a simple strategy for the selection of antisense fragment targeting human genes.更多还原