作　　者： ; ; ; ; ; ; ;

机构地区： 中国农业科学院北京畜牧兽医研究所

出　　处： 《广西农业生物科学》 2006年第B09期182-183,共2页

摘　　要： 2005年我国获得了体细胞克隆猪的成功,但是在体细胞转基因克隆猪方面还处于起步阶段。猪的转基因克隆在农业和医学方面有着巨大的应用前景,在农业上可以提高猪肉品质和培育抗病猪种等;医学上可以为人类异种器官移植、疾病模型和新药筛选提供理想材料。体细胞核移植结合基因组修饰技术,是目前生产转基因家畜的一种有效方式。供体细胞的类型是影响转基因克隆效率的一个关键因素,因为供体细胞的类型决定着体细胞转基因效率和重组胚的发育潜力。目前所知,较成功用于生产转基因克隆猪的供体细胞仅限于胎儿成纤维细胞,因为它易培养、增殖期限长、重构胚的发育能力高。此外骨髓间充质细胞,一种成体干细胞,作为转基因克隆猪的供体细胞有着巨大的潜力,国内外仅有个别报道。前脂肪细胞,一种易于获得的体细胞,有报道称利用成年猪前脂肪细胞为核供体构建的克隆胚发育能力和胎儿成纤维细胞相当,并且获得了克隆猪的后代。而利用猪前脂肪细胞生产转基因克隆胚胎,将为后续组织工程研究奠定基础。因此本研究选择猪胎儿成纤维细胞、骨髓间充质细胞和前脂肪细胞为转基因克隆的供体细胞,目的是比较不同类型转基因供体细胞对生产克隆胚胎效率的影响。本研究首先建立了近交系五指山小型猪(WZSP)胎儿成纤维细胞系(FF)、新生猪骨髓间充质细胞(MSCs)系和成年猪前脂肪细胞系(PreA)。采用组织块法建立胎儿(27日龄)成纤维细胞系;髓间充质细胞从新生猪后腿骨冲取骨髓,加入到Percoll分离液中离心,吸取中间界层面细胞,贴壁法培养骨髓间充质原代细胞,培养基为低糖DMEM。对间充质细胞向脂肪细胞进行诱导分化,油红O染色鉴定;前脂肪细胞从猪的皮下脂肪取样,用0.1%胶原酶消化法建立前脂肪细胞原代细胞,并对前脂肪细胞进行诱导分化为成� Somatic cell nuclear transfer （SCNT）, combined with genome modification techniques, is a very powerful tool for agriculture, medicine and fundamental research on basic biological mechanisms. The present study examined the effects of different types of transgenic donor cells on developmental potential of porcine nuclear transfer （NT） embryos. Three types of somatic cells from Inbred Miniature Wuzhishan Pig （WZSP）, including fetal fibroblasts （FF）, bone marrow mesenchymal stem cells （ MSCs ） , and adult preadipocytes （ PreA ） were transfered with a plasmid pEGFP - N 1 by using a lipofectamineTM 2 0 0 0 （ invitrogene , Life Technologies ） . After 1 4 days selection with 800μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic and transgenic cells were used as nuclear donor to produce NT embryos. The results were as follows： （1） There was no significant difference in development rates to the blastocyst stage for NT embryos among three different types （FF,MSC and PreA） of nontransgenic donor cells （8.2 %, 7.1% vs 8.8%, P〈0.05）. （2） The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells （P〈0.05）. Blastocyst rates from FF and MSC cells （9.6% and 9.9 %, respectively） were higher than those from PreA cells （3.7 % ）; （3） There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic FF cells （9.6% vs 8.2%）, MSC cells （ 9.9% vs 7.1%） respectively （P〉0.05）. However, using PreA cells expressing green fluorescent protein （GFP） as donors, the blastocyst rate of cloned embryos decreased drastically in comparison to those derived from nontransgenic PreA （3.7% vs 8.8%, P〈0.05） . These results suggest that FF and MSC cells can be used to produce transgenic porcine NT embryos more efficiently. Further study needs to be done to observation that efficiency of

关 键 词： 猪 体细胞 核移植 绿色荧光蛋白 转基因 囊胚

领　　域： [农业科学] [农业科学]