实时荧光定量PCR检测转基因小鼠外源基因拷贝数方法的建立和应用  

Establishment and application of the method for detecting copy number of foreign gene in transgenic mice with Real-Time Fluorescent Quantitative PCR

作　　者：郑杰辉[1] 林炤华[1] 徐瑛[1] 刘军[1] 周常文[1] 

机构地区：[1]福建医科大学细胞生物学与遗传学系细胞与发育工程研究中心,福州350004

出　　处：《中国优生与遗传杂志》2011年第2期34-36,80,共4页Chinese Journal of Birth Health & Heredity

基　　金：福建医科大学科研发展基金(XZ04005)

摘　　要：目的建立实时荧光定量PCR方法检测CaMKⅡα-Cre转基因小鼠外源基因拷贝数的方法。方法以CaMKⅡα-Cre转基因首建鼠及其仔代阳性鼠为研究对象,利用绝对定量的实时荧光定量PCR法检测转基因小鼠的拷贝数,并筛选出纯合子小鼠再经遗传育种方法以确定为纯合子小鼠。结果绝对定量标准曲线公式为:△Ct=-2.402log5N(拷贝数)+8.654,相关系数为0.9999,扩增效率为95.4%。三只CaMKⅡα-Cre首建鼠的拷贝数分别为19、7、5;三个转基因小鼠品系均成功获得纯合子小鼠。结论成功建立了检测转基因小鼠外源基因拷贝数的实时荧光定量PCR方法,该方法可用于检测各种转基因小鼠中外源基因的拷贝数。Objective：To develop a novel method to determine transgene copy number in Genome of CaMKⅡα-Cre Transgenic Mice by using the real-time fluorescent quantitative PCR.Methods：The CaMKⅡα-Cre founder mice and their offsprings were studied.The transgene copy numbers in transgenic mice were determined by real-time fluorescent quantitative PCR,then the homozygote mice was obtained and proved by genetic breeding.Results：The equation of absolute quantitative standard curve was △Ct =-2.402log5N ＋ 8.654.The correlation coefficient was 0.9999,E=95.4%.Three CaMKⅡα-Cre founders were detected to have the transgene copies of 19,7,5,respectively.The homozygote transgenic mice were obtained successfully.Conclusion：A real-time fluorescent quantitative PCR method was successfully established,which might be used for determining transgene copy number in various transgenic mice.

关 键 词：转基因小鼠 拷贝数 实时荧光定量PCR SYBRGreenⅠ CT值 

分 类 号：R346[医药卫生—基础医学]