实时荧光定量PCR法检测转基因小鼠拷贝数

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。

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

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.