核基质附着区(MAR)调控的人胰岛素样生长因子(hIGF-1)转基因兔的制备

Production of Transgenic Rabbit with hIGF1 cDNA Regulated by Matrix Attachment Region

用ＥｃｏＲＩ＋ＨｉｎｄⅢ双酶解含鸡α－珠蛋白基因５′端核基质附着区（ＭＡＲ）、小鼠金属硫蛋白基因启动子（ＭＴ－１）和人胰岛素样生长因子－１（ｈＩＧＦ－１）基因的ｐＭＴＳＭＣＡＧ质粒，０．６％琼脂糖凝胶电泳，玻璃乳回收纯化ＭＡＲ／ＭＴ／ｈＩＧＦ－１片段（３．９ｋｂ），用该片段制备转基因兔。利用水平显微注射系统注射１－细胞兔胚胎４６８枚，移植到３６只受体兔，有８只妊娠，共产下１９只活仔兔。采仔兔耳样提取基因组ＤＮＡ，应用ＰＣＲ技术分析其外源基因整合情况，获得８只阳性兔（显示出约６００ｂｐ外源ＤＮＡ条带）。再对８只阳性兔的ＰＣＲ产物进行Ｓｏｕｔｈｅｒｎ印迹杂交，得到５只整合有ＭＡＲ／ＭＴ／ｈＩＧＦ－１外源基因的转基因兔，转基因整合率为２６．３％（５／１９）。用１只阳性转基因公兔（５０２号）繁殖了６窝，共获得４２只仔兔。采仔兔耳样提取基因组ＤＮＡ后，如上用ＰＣＲ分析和做Ｓｏｕｔｈｅｒｎ印迹杂交，Ｆ１代中有５只整合有ＭＡＲ／ＭＴ／ｈＩＧＦ－１融合基因。

A 3.9 kb fragment digested with Eco RI and Hind Ⅲ was isolated from agarose Gel using Glass MAXMatrix isolation kits. This fragment, which consists of the chickenalpha globin gene(5′MAR), mousemetallothionein1 (promoter) and human insulinlike growth factor1, was microinjected into male pronuclei of 468 rabbit 1cell embryos, and injected embryos were transferred into the oviduct of 36 recipients. Eight rabbits of 36 recipients were pregnant and 19 live offsprings born. Genomic DNA was extracted from ear specimens of rabbits (F0) and amplified with a pair of primerMP1 and MP2 by polymerase chain reaction to analyze the integration of foreign gene. A specific band of 600 bp foreign DNA present in eight transgenic positive rabbits through PCR screening. Five of eight positive rabbits proved to be transgenic rabbit for MAR/MT/hIGF1 fusion gene based on Southern hybridization analysis, giving an integration rate of 26.3% (5/19). 42 young (F1) were generated by one male transgenic rabbit (number 502) servicing 6 nontransgenic female rabbits. PCR products from young′s genomic DNA identified by southern blot indicated that five of 42 F1 rabbits were transgenic rabbits integrated MAR/MT/hIGF1.