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cDNA Cloning of Goat DNA Methyltransferase 1,Screening of shRNA Vectors and Influences to Development of Nuclear Transfer Embryos
Authors:LAN Jie  SONG Yong-li  HUA Song  LIU Yong-gang  LIU Jun  ZHANG Hai-lin  ZHANG Yong
Institution:1. Department of Cell and Molecular Physiology, Loyola University Chicago Health Science Division, Maywood, Illinois;1. Unidad de Genética, Grupo GENIUROS, Escuela de Medicina y Ciencias de la Salud, Universidad del Rosario, Bogota, Colombia;2. Unidad de Fertilidad PMA, Clínica Marly, Bogota, Colombia;1. State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, P.R. China;2. State Key Laboratory for Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R. China;1. College of Animal Science & Technology, Shaan’xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, Yangling, Shaan’xi 712100, China;2. Research Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China;1. The First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210046, Jiangsu Province, China;2. Department of Oncology, Nantong Hospital of Traditional Chinese Medicine, Nanjing University of Chinese Medicine, Nantong 226000, Jiangsu Province, China;3. Department of Respiratory Medicine, Nantong Third People''s Hospital, Nantong 226006, Jiangsu Province, China
Abstract:This study was designed to clone cDNA of goat DNA methyltransferase 1(DNMT1)gene,to screen an effective shRNA-producing 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 methyitransferase 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.
Keywords:cDNA  DNA methyltransferase 1  goat  methylation  shRNA
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