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为了在转基因猪肌肉中高效率表达Fat-1基因,且进一步得到富含ω-3 PUFAs猪品系建立技术基础。将化学合成的人源化Fat-1基因克隆到表达载体上,构建能够在猪细胞中高效表达的质粒构件p MSTN-ω3,使用胚胎原核显微注射方法制备转h Fat-1基因猪。试验对16头供体母猪超数排卵,人工授精配种后手术法冲取320枚1~2细胞期猪胚胎。原核显微注射法向胚胎导入线性化质粒p MSTN-ω3,302枚1~2细胞期胚胎显微注射后移植10头受体母猪,5头母猪妊娠,生产26头仔猪。PCR法检测得到4头转h Fat-1基因阳性猪,Southern印迹杂交分析验证1头猪整合有h Fat-1基因,1头猪近似整合h Fat-1基因。有1头h Fat-1基因整合猪可用于转基因猪育种,表达构件p MSTN-ω3能够用于生产转Fat-1基因猪。试验成功采用显微注射法制备转h Fat-1基因猪,改进、规范了胚胎原核显微注射法制备转基因猪的试验操作程序。
To establish a technical basis for the efficient expression of the Fat-1 gene in the transgenic pig muscle and to further obtain pig lines rich in omega-3 PUFAs. The chemically synthesized humanized Fat-1 gene was cloned into the expression vector to construct a plasmid p MSTN-ω3 capable of being highly expressed in porcine cells. The embryo pronucleus microinjection method was used to prepare h-Fat-1 transgenic pigs. Twenty-six donor sows were superovulated, and 320 1 to 2-cell pig embryos were scored by surgery after artificial insemination. Progenitor microinjection into the embryo into the linearized plasmid p MSTN-ω3,302 1 ~ 2 cell stage embryos after microinjection 10 recipient sows, 5 sows pregnant, producing 26 piglets. Four pigs positive for h-Fat-1 gene were detected by PCR, and h Fat-1 gene was integrated into one pig by Southern blotting analysis. One pig was approximately integrated with h-Fat-1 gene. One h Fat-1 gene integrated pig was used for transgenic pig breeding and the expression construct p MSTN-ω3 could be used to produce transgenic pig Fat-1. Experiments successfully using microinjection method to prepare h-Fat-1 transgenic pigs, improve and standardize embryo pronucleus micro-injection method for preparing transgenic pig experimental procedures.