International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA


POSTER 136 - USING REAL-TIME PCR ALLELIC DISCRIMINATION ASSAY TO QUANTITATE TRANSGENES IN PHENOTYPE RESCUE EXPERIMENTS

Bosak NP, Li S, Li X, Reed DR, Beauchamp GK, Bachmanov AA

Monell Chemical Senses Center, Philadelphia, United States

To determine the zygosity of transgenic animals and transgene copy number with currently available techniques demands tedious procedures with sometimes ambiguous results. Real-time PCR is a high throughput quantitative method, accurate and sensitive enough to discriminate a two-fold difference in the amount of target sequences. We investigated the validity of allelic discrimination assay using real-time PCR with allele-specific probes for the transgene quantification. We used DBA/2J;129P3/J-TgN(Tas1r3B6Ge)41Mon mice obtained in a transgenic complementation (phenotype rescue) experiment. The transgene-positive mice were either homozygous or hemizygous for the transgene. The assay was optimized to distinguish between a transgenic and an endogenous allele of the Tas1r3 gene. We used two quantitation approaches: 1) the comparative cycle threshold method and 2) endpoint reading of normalized fluorescence.  Zygosity status was determined using the allelic discrimination assay, and was confirmed by Southern analysis and progeny segregation. Real-time quantitative PCR allows precise quantification of transgene copy number and rapid determination of zygosity in transgenic animals. Furthermore it is more efficient than the traditional methods and particularly suitable for phenotype rescue transgenic experiments.

Supported by NIH grants R01AA11028 (AAB) and R01DC00882 (GKB).

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