[A simple strategy for the rapid selection of effective antisense fragments targeting human genes].
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.
Li
Fen
F
College of Life Science, Henan Normal University, Xinxiang 453007, China. lifen2001@yahoo.com
Ma
Ji Xian
JX
Bai
Xue
X
Chen
Bai Bao
BB
D016373:DNA, Antisense / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D016373:DNA, Antisense; D009419:Nerve Tissue Proteins; D029701:Saccharomyces cerevisiae Proteins; C538729:ELP3 protein, human; C526363:Elp3 protein, S cerevisiae; D051548:Histone Acetyltransferases