利用实时荧光定量比较Ct法检测转基因小鼠外源基因拷贝数  

Estimation of the Copy Numbers of Exogenous Gene in Transgenic Mice Using the Real-Time Fluorescence Quantitative PCR Based Comparative Ct Method

作　　者：林维石[1] 王芃[1] 程萱[1] 袁盛凌[1] 陈红星[1] 林艳丽[1] 陶好霞[1] 王艳春[1] 王令春[1] 刘纯杰[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2013年第4期497-500,共4页Letters in Biotechnology

基　　金：国家重大新药创制项目(2012ZX09301003-001-005)

摘　　要：目的:在建立转基因小鼠模型时,外源基因拷贝数是影响其表达水平和遗传稳定性的重要因素之一。外源基因拷贝数的精确测定,是建立转基因动物模型的重要环节。方法:合成cagA基因和内参基因GAPDH的引物,用标准曲线法测得cagA和GAPDH基因的扩增效率分别为97.6%和98.6%;将128拷贝阴性小鼠基因组和128拷贝cagA打靶质粒的混合物作为参照样品,取6只来自同一母本的F2阳性小鼠的128拷贝基因组作为待测样品;选取GAPDH作为内源参照基因,用比较Ct法对待测样品进行定量。结果:经计算,6只待测小鼠的cagA基因拷贝数平均值为8。结论:利用实时荧光定量PCR仪,采用改良后的比较Ct法对转基因小鼠的外源基因拷贝数进行了精确测定。Objective： The copy number of exogenous gene was an important factor that greatly influenced the ex- pression level and genetic stability of the target gene in transgenie mice model. Estimating the transgene copy numbers becomes a significant step in a transgenic research. Methods： The primers of cagA and GAPDH were synthesized, and standard curve analysis showed that the amplification efficiency of cagA and GAPDH were 97.6% and 98.6% respectively. 128 copies of wild-type mice genomic DNA and 128 copies of cagA transgenic plasmids were mixed as reference samples, while 128 copies of genomic DNA from each of six F2 generation cagA transgen- ic mice were used as samples under test. The GAPDH gene was used as endogenous reference gene. The copy number was detected by the comparative Ct method. Results： The mean copy number of the cagA transgenic mice is 8 in this study. Conclusion： With a real time fluorescence quantitative PCR instrument, we made a precise cal-culation of exogenous ~ene number of the transgenie mice by an improved comparative Ct method.

关 键 词：转基因小鼠 拷贝数 比较Ct法 实时荧光定量PCR 

分 类 号：Q78[生物学—分子生物学]