Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR.

Abstract

Successful identification of homozygous and heterozygous transgenic animals with currently available techniques demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large numbers of animals differing only by a factor of two in the amount of target sequences. We defined the technical conditions of real-time PCR to co-amplify a transgene and a reference gene using two fluorogenic probes and the comparative cycle threshold method. We applied these conditions to the analysis of zygosity in a line of transgenic rats. Real-time PCR allowed clear-cut identification of all transgenic animals analysed (n = 45) as homozygous or heterozygous. Southern blot analysis of these animals using an internal quantitative control and PhosphorImager quantification showed ambiguous results in six of them and was concordant with real-time PCR in the rest. Mating of homozygous and heterozygous animals, as defined by real-time PCR, showed transgene transmission to the offspring following expected Mendelian laws. Real-time PCR allows rapid, precise, non-ambiguous and high throughput identification of zygosity in transgenic animals. This technique could be helpful in the establishment of breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact.