转K2.9基因绒山羊体细胞核移植技术体系的优化研究

【目的】利用体细胞核移植技术制备转毛角蛋白Ⅱ型中间丝K2.9基因的高绒质绒山羊胚胎，为绒山羊优良品种的培育提供一种全新的技术材料。【方法】以含有Neor 基因标记的K2.9 毛囊特异表达载体pcDNA3.1-K转染绒山羊胎儿成纤维细胞，经G418筛选获得转K2.9基因细胞，将获得的转基因阳性细胞与体外成熟的绒山羊卵母细胞进行核移植，并对生产的重构胚进行了体外培养。本文分别进行了激活方法、供体细胞和卵母细胞来源的筛选，且对获得的囊胚进行了PCR鉴定。【结果】（1）Iono+6-D对成年羊卵母细胞的孤雌激活效果好于A23187+6-D，显著提高了胚胎的卵裂率。（2）羔羊孤雌胚的卵裂率显著低于成年羊，但囊胚率差异不显著。（3）来自2只绒山羊胎儿的转基因成纤维细胞对核移植胚的发育没有显著影响，但2号羊的转基因细胞显著提高了融合率。（4）以羔羊卵进行核移植，显著降低了核移植胚的发育率。（5）对获得的囊胚进行PCR鉴定，成功扩增到目的基因。【结论】将K2.9 毛囊特异表达载体pcDNA3.1-K转染的绒山羊胎儿成纤维细胞作为供体细胞，核移植到成年羊卵母细胞中，以Iono+6-D进行激活，首次成功、高效地获得携带K2.9基因的绒山羊囊胚。

Construction of Cashmere Goat Embryos Carrying K2.9 Gene by Transgenic Somatic Cell Nuclear Transfer Technology

【Objective】In order to change the protein composition of wool and improve the quality of wool,transgenic somatic cell nuclear transfer technology was used to prepare K2.9(gene from hair keratin intermediate filament type Ⅱ) transgenic cashmere goat embryos to produce transgenic goats.【Method】 Cashmere goat fetal fibroblast cells were transfected by hair follice-specific expression vector pcDNA3.1-K containing K2.9 and Neor gene,and G418 was used to select transgenic cells as donor cells.To improve the efficiency of nuclear transfer technology,the effects of the activation methods and the sources of donor cells and oocytes on the development of parthenogenetic embryos were studied.Identification of the genomic DNA of transgenic blastocysts by ploymerase chain reaction(PCR) has proved that the exogenous gene had already been integrated into genomes of blastocyst cells.【Result】 The cleavage rate of adult cashmere goat parthenogenetic embryos was significantly increased by Iono+6-D activation.Iono+6-D was more suitable to the activation of adult cashmere goat oocytes than A23187+6-D.The cleavage rate of lamb pathenogenetic embryos was significantly lower than that of adult cashmere goat parthenogenetic embryos,but the blastocyst rate had no significant difference.The development of nuclear transfer embryos from the transgenic fibroblast cells of two cashmere goat fetuses as donor cells weren’t affected,but the fusion rate of nuclear transfer embryos from the transgenic fibroblast cells of 2nd cashmere goat fetuse as donor cells were greatly increased.The development of nuclear transfer embryos from lamb oocytes was signicicantly reduced.The manipulated blastocysts were confirmed carrying K2.9 gene by PCR.【Conclusion】The transgenic somatic cell nuclear transfer technology could produce cashmere goat blastocysts carrying K2.9 gene,which were constructed through K2.9 transgenic cashmere goat fetal fibroblast cells by hair follicle-specific expression vector pcDNA3.1-K as donor cells and adult cashmere goat oocytes as receptors,and activated by Iono+6-D.