摘要
种蛋内鸡胚含有潜在的胚胎干细胞(BCs)或原生殖细胞(PGCs),是目前主要的转基因鸡研究方法。采用绿色荧光蛋白(GFP)基因的pLenti6/v5-DEST-GFP慢病毒表达载体,白来航鸡与伊萨鸡种蛋,结合种蛋赤道面开窗专利技术,对含这两种细胞胚的转基因技术进行了比较研究:转染白来航鸡囊胚,孵化13天时,GFP基因的PCR检出率为64.7%,孵化率极低;转染孵化72h伊萨鸡胚血液循环中PGCs,实验蛋孵化率为35.0%,在出壳后死亡的3只小鸡肝脏中,GFP基因的PCR检出率为100%,存活的4只鸡中有3只在12月龄的血液样品中,经PCR扩增出了GFP基因;转染孵化72～79h白来航鸡胚PGCs,7批次实验的平均孵化率为21.1%,能在赤道面窗口注射胚的种蛋比率,以73～77h胚龄的最高,为75.0%～92.9%,注射病毒组出壳雏鸡血液DNA中,GFP基因PCR检出率为44.4%。两种方法比较,PGCs方法在实验蛋孵化率、胚定位在赤道面窗口率等方面有较强优势。因此为种蛋内胚细胞的转基因鸡技术研究提供了系统、可操作性强的方法。
There were two kinds of pluripotent stem cells in the fertile chicken eggs, one was blastodermal cells （BCs） and the other was primordial germ ceils （PGCs） and both of them were the main methods of transgenic chicken. The White Leghorn （WL） and Isa Brown （IB） fertile eggs at the stage of BCs and PGCs, were windowed equator/ally, and the Lentivr/al vector pLenti6/vS-DEST-EGFP was microinjected into the eggs. For BCs fertile eggs from WL, the positive embryos was 64.7% （11/17）when the 13-day embryos were checked by PCR. For the PGCs （ eggs incubated for72h） stage fertile eggs from IB, the hatchability was 35%, three chickens died little time after hatching, which were all positive when checked using the liver DNA by PCR at the age of 1 month and 6 month. There were three chickens among four alive ones （75%） was positive when checked the blood DNA by PCR, however they were very weak positive at the age of 12 month. For the PGCs （eggs incubated for 72 - 79h） stage fertile eggs from WL, the average hatehability was 21. 1% , the ratio of being able to injected eggs through the window was 75. 0% - 92. 9%, which was highest and covered the incubated time from 73h to 77h. The ratio was 44.4% when the hatched chickens were checked using the blood DNA by PCR. Comparing BCs method, the PGCs method was much better, such as on the hatchability, embryo locating on the equatorial window and so on.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2012年第12期73-79,共7页
China Biotechnology
基金
国际科技合作计划(中方2010-96-39-1)
捷克教育部资金(No.10104)资助项目
关键词
慢病毒载体
种蛋
胚
Lentivirus vector Fertile eggs Embryos Transgenic chicken
