摘要
目的:利用real-time PCR建立检测C57-ras转基因小鼠中外源c-Ha-ras基因拷贝数的简便方法,为药物安全性评价C57-ras转基因小鼠模型的繁殖和筛选提供数据支持。方法:以自建的人原癌基因c-Ha-ras转基因小鼠为研究对象,利用SYBR GreenⅠ荧光定量PCR的绝对定量法测定3个C57-ras转基因小鼠系的c-Ha-ras基因Ct值,通过与内参基因GAPDH比较计算获得转基因的拷贝数。结果:内参基因GAPDH的标准曲线为lg NGAPDH=-2.852Ct+26.236,外源基因c-Ha-ras的标准曲线为lg NRAS=-3.068Ct+39.186;经计算,NO.2、NO.3、NO.5系的F6代拷贝数分别为4、4和3,并且系内不同个体间拷贝数一致。结论:利用SYBR GreenⅠreal-time PCR技术建立了检测C57-ras转基因小鼠中外源基因拷贝数的方法,该方法操作简单,节约成本,为C57-ras致癌性模型的选留和应用提供了基础和技术手段,也为其他类似转基因品系中转基因拷贝数的确定提供了一种参考方法。
Objective： To establish a time and cost saving method for analyzing copy number of transgene in C57-ras transgenic mice line breeding and screening. Methods： Three C57-ras transgenic mice founder lines, which were inserted with human prototype c-Ha-ras gene, were derived and detected for copy number analysis. The Ct value of transgene c-Ha-ras and internal gene GAPDH were detected by SYBR GreenⅠreal-time absolute quantitative PCR method, and the copy number were obtained by calculating the ratio c-Ha-ras/GAPDH. Results：The equation of absolute quantitative standard curve of GAPDH, as internal reference gene, was lgNGAPDH=-2.852Ct＋26.236, while the equation of absolute quantitative standard curve of c-Ha-ras was lgNRAS=-3.068Ct＋39.186. The transgene copies of three C57-ras trangenic mice lines were 4, 4 and 3, respectively. Conclusion： SYBR GreenⅠreal-time absolute quantitative PCR method, which is easy and time-saving, was successfully established for transgene copy number detection in C57-ras mice. This valid copy number analysis method will help us to screen the most appropriate transgenic lines for carcinogenicity tests, and may be used as a candidate method for other transgenic line selections.
出处
《生物技术通讯》
CAS
2015年第2期227-231,共5页
Letters in Biotechnology
