cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos

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 

作者机构：Key Laboratory of Animal Reproductive Physiology & Embryo Technology Institution of Biotechnology College of Veterinary Medicine Northwest A&F University Yangling 712100 P.R. China 

出 版 物：《Agricultural Sciences in China》 (中国农业科学（英文版）)

年 卷 期：2010年第9卷第7期

页      面：1035-1040页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：funded by the Key Scientific and Technological Special Program of China (2008ZX08007004) 

主　　题：cDNA DNA methyltransferase 1 goat methylation shRNA 

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 *** 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 ***,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 *** the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic ***,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 *** results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned（GenBank ***617538）.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.