构建转CgA基因反向cDNA片段小鼠模型  

Construction of CgA Gene Antisense Transgenic Mice

作　　者：刘晓东[1] 孙紫清[1] 张崇本[1] 吴鹤龄[1] 

机构地区：[1]北京大学生命科学学院,100871

出　　处：《Acta Genetica Sinica》2001年第6期493-501,共9页

基　　金：国家自然科学基金资助(资助号.39470371)&&

摘　　要：为了制作ＣｇＡ基因反义ＲＮＡ转基因小鼠，构建了质粒ｐＣＡＳ２Ｃ，并把ｐＣＡＳ２Ｃ中完整 的反义表达框显微注射入小鼠受精卵的雌原核中，随后将注射过ＤＮＡ的受精卵移植入假 孕鼠的输卵管中。假母所产子一代都用ｐ１和ｐ４引物对鼠尾基因组ＤＮＡ进行ＰＣＲ检测，选 出２只首建鼠（ＰＣＲ阳性）。首建鼠与正常鼠杂交后得到Ｆ１代小鼠，经ＰＣＲ方法筛选出来的 阳性Ｆ１代鼠雌雄自交产生Ｆ２代。为了检测Ｆ２代鼠的基因型，将ＰＣＲ方法鉴定为阳性的 Ｆ２代 小鼠分别与正常小鼠回交。只有回交后所得后代ＰＣＲ检测结果全为阳性的Ｆ２代个体才是 转基因纯合鼠。这样分别得到两只转基因首建鼠的纯合个体。转基因小鼠脑总ＲＮＡ经 ＲＴ－ＰＣＲ反应后可产生３００ｂｐ的产物，证明转基因小鼠中转入的反义基因已经转录。以上结 果说明转入的ｐＣＡＳ２Ｃ反义表达框已经整合到转基因小鼠的基因组中并遗传给后代，而且 已经转录。In order to get CgA gene antisense DNA transgenic mouse, we constructed the CgA gene antisense DNA plasmid pCAS2C and microinjected it into the female pronucleus of fertilized mouse eggs, and transplanted them into oviduct of the foster. Every offspring of the fosters was determined by the PCR method. The positive mice had a 300bp DNA eletrophoresis band. We selected two male positive mice from 50 offspring survived of the pseudomother. Then, two positive mice crossed with normal mice respectively to reproduce offspring of F1. All offspring of F1 were deterdmined by PCR to select positive offspring. Positive offspring of F1 carried only one allele of pCAS2C (heterozygous pCAS2C / -). Positive F1 offspring were selfcrossed, 1/ 4 offspring of F2 carrying two unites of one allele pCAS2C (pCAS2C / pCAS2C) are homozygous. Then, all offspring of homozygous F2 crossed with normal mice, could Produce 300bp DNA electrophoresis band by PCK Total RNA of brain tissue of transgenic mouse was used to RT-MCR method, the 300bp DNA product was obtained. The result indicates that the reading frame of CgA antisense DNA of pCAS2C has expressed in the transgenic mice.

关 键 词：CgA基因 反义RNA 转基因小鼠 cDNA片断 

分 类 号：Q754[生物学—分子生物学]