摘要
This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.
This study was designed to clone cDNA of goat DNA methyltransferase 1（DNMT1） gene,to screen an effective shRNAproducing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells.In this study,PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions.Next,3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector,acquiring 3 new vectors,respectively termed pRNAD1,pRNAD2 and pRNAD3.Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing.Finally,the developmental rates of nuclear transfer（NT） embryos generated using donor cells with and without the effective shRNA vector respectively,as well as in vitro fertilization（IVF） embryos were observed and recorded.The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank no.FJ617538）.Furthermore,an effective interfering shRNA（pRNAD2） was obtained,with its interference effect being 47.88%.Finally,NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls（P 〈 0.05）,reaching the similar rates to IVF embryos（P 〉 0.05）.In conclusion,goat DNA methyltransferase 1 gene cDNA was cloned and sequenced,an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved,which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.
作者
LAN Jie,SONG Yong-li,HUA Song,LIU Yong-gang,LIU Jun,ZHANG Hai-lin and ZHANG Yong Key Laboratory of Animal Reproductive Physiology & Embryo Technology,Institution of Biotechnology,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,P.R.China
基金
funded by the Key Scientific and Technological Special Program of China (2008ZX08007004)
