实时荧光定量PCR法检测转基因小鼠拷贝数  

Absolute Quantitative Real-Time PCR Assay for Determining Transgene Copy Number in Transgenic Mice

作　　者：王晓建[1] 杨旭[1] 宋晓东[1] 张禅那[1] 刘继斌[1] 邸冉[2] 张连峰[2] 惠汝太 

机构地区：[1]中国医学科学院阜外心血管病医院心血管病相关基因与临床研究教育部重点实验室(中德实验室),北京100037 [2]中国医学科学院实验动物研究所分子遗传中心,北京100021

出　　处：《中国实验动物学报》2007年第3期170-174,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：北京市自然科学基金重大项目(编号:04G409)

摘　　要：目的利用实时荧光定量PCR法鉴定转基因小鼠外源基因插入拷贝数。方法以TG-CARK转基因首见鼠为研究对象,选取小鼠的高度保守基因Fabpi为内参,利用绝对定量的实时荧光PCR法鉴定转基因小鼠拷贝数,并与传统的Southern blot方法的定量结果进行比较。结果实时定量PCR鉴定的转基因拷贝数与Southernblot法完全一致,三只TG-CARK首见小鼠的拷贝数分别为1,7,45。结论实时定量PCR技术具有高准确性、高稳定性、高通量和低成本的优点,是比传统杂交技术更好的鉴定小鼠转基因拷贝数的方法。Objective It is first and foremost to genotype and determine the transgene copy number for making a transgenic model. Southern blot analysis, the traditional means, is time consuming and laborious. The aim of this study was to built up a novel method to determine transgene copy number by using the absolute quantitative real-time PCR. Methods Taken TG-CARK founder mice as examples, the transgene copy numbers in transgenic mice were determined by quantitative real-time PCR and Southern blot. The conserved mouse housekeeping gene, Fabpi, served as an internal control to calculate transgene copy number. Results The copy numbers of transgenic founders estimated by real-time quantitative PCR were highly correlated with actual copy number based on Southern blot assay. The three TG-CARK founders had the transgene copies of 1, 7, 45 respectively. Conclusion Real-time PCR is a more efficient and convenient method for estimating copy number in transgenic mice than Southern blot.

关 键 词：转基因 拷贝数 实时定量PCR 

分 类 号：R331[医药卫生—人体生理学]