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

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 transgenic 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 gene number of the transgenic mice by an improved comparative Ct method.