农业生物技术学报2011，Vol.19Issue(2)：294-301,8.DOI:10.3969/j.issn.1674-7968.2011.02.014
体细胞核移植技术生产转入溶菌酶基因(hLYZ)牛胚胎的研究
Human Lysozyme Gene (hLYZ) Transgenic Bovine Embryos Produced by Somatic Cell Nuclear Transfer
摘要
Abstract
The aim of this study was to investigate the effect of different sizes and treatments of donor cells on the in vitro development of bovine embryos after genetic modification and selection, so as to establish an effective system for production of transgenic bovine embryos.Bovine fetal fibroblasts were transfeected with the recombinant plasmid of mammary gland specific expression vector pEBH which containing human lysozyme (hLYZ) gene.Transgenic positive cells which were obtained through G418 selection were used as donor cells for production of transgenic cloned embryos.The result showed that genetic modification and screening of donor cells were harmful for the development of transgenic cloned embryos, the blastocyst rate decreased significantly;referred to internal diameter of injective needle, cells with the diameter of 15～20 μm were selected and used as nuclear transfer donor cells, the fusion rate and blastocyst rate of transgenic cloned embryos were significant higher than that of the other groups (P ＜ 0.05); Compared with serum starvation and contact restrain groups, the cleavage rate and blastocyst rate of transgenic cloned embryos which were constructed with plant cells were significantly higher (P ＜ 0.05); The expression of GFP was observed in each stage of in vitro development in all transgenic cloned embryos, but the expression levels seemed to be vary among individuals and even among different developmental stages of the same individual.We chose I 0 embroys randomly for PCR detection and found that all of the embroys were positive.Above all, we obtained hLYZ transgenic cloned embryos by somatic cell nuclear transfer technique, the reconstructed embryos can develop to blastocysts successfully; and when plant cells with diameter of 15～20 μm were used as donor cell, the developmental competence of somatic cell cloned embryos is improved significantly.关键词
人溶菌酶/转基因/供体细胞/核移植/牛Key words
Human lysozyme/ Transgene/ Donor cells/ Nuclear transfer/ Bovine引用本文复制引用
熊显荣,李文哲,王丽君,王勇胜,苏建民,华松,张涌..体细胞核移植技术生产转入溶菌酶基因(hLYZ)牛胚胎的研究[J].农业生物技术学报,2011,19(2):294-301,8.基金项目
本研究由国家科技部抗病转基因牛新品种培育项目(No.2008ZX08007-004)资助 （No.2008ZX08007-004）
