c-Ha-ras转基因小鼠的构建与鉴定

Construction and Identify of c-Ha-ras Transgenic Mice

采用 ＰＣＲ和定点突变的方法获得 ｃ－Ｈａ－ｒａｓ突变基因，将其克隆到ｐｃＤＮＡ３载体上，得到含有 ｃ Ｈａ－ｒａｓ６１密码子突变和核苷酸 ２７１３突变的质粒 ｐｃＤＮＡ３－ｒａｓ２。对重组质粒进行瞬时表达，Ｗｅｓｔｅｒｎ Ｂｌｏｔ检测到强阳性条带。酶切重组质粒，回收含有ＣＭＶ启动子－目的基因－下游调控序列的４．３ｋｂ片段，然后将其显微注射到１ＣＲ小鼠受精卵中。将注射后的受精卵移入假孕母鼠的输卵管进行母体孕育。小鼠出生３周后，ＰＣＲ和Ｓｏｕｔｈ－ｅｒｎ Ｂｌｏｔ检测证实有４只小鼠基因组中整合了外源ｃ－Ｈａ－ｒａｓ突变基因。上述结果表明已成功构建了 ４只 ｃ－Ｈａ－ｒａｓ转基因小鼠。

The c-Ha-ras gene was obtained by PCR and site-specific mutation, then cloned them to vector pcD-NA3. We got pcDNA3-ras2 plasmid, containing the mutation of 61 coden and nucleotide 2713. We carried out the transient expression experiment of the recombinated plasmid and got strong positive band by Western Blot. Then we recovered the 4.3 kb fragment containing the upstream CMV promoter, c-Ha-ras gene and downstream regulor reagion. The fragment was microinjected to the fertilized eggs of ICR mice, then the fer-tilized eggs were transplanted into the oviduct of the psedu-pregnant mice to gestate. After the mice were born, PCR and Southern Blot exipriment proved that the DNA of four mice have integrated with the trans-gene. The result showed that we have succeeded in the construction of c-Ha-ras transgenice mice.