CAJ | 학술논문

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。
목적：재건립전기인소서모형시，외원기인고패수시영향기표체수평화유전은정성적중요인소지일。외원기인고패수적정학측정，시건립전기인동물모형적중요배절。방법：합성cagA기인화내삼기인GAPDH적인물，용표준곡선법측득cagA화GAPDH기인적확증효솔분별위97.6%화98.6%；장128고패음성소서기인조화128고패cagA타파질립적혼합물작위삼조양품，취6지래자동일모본적F2양성소서적128고패기인조작위대측양품；선취GAPDH작위내원삼조기인，용비교Ct법대대측양품진행정량。결과：경계산，6지대측소서적cagA기인고패수평균치위8。결론：이용실시형광정량PCR의，채용개량후적비교Ct법대전기인소서적외원기인고패수진행료정학측정。
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.