RT-PCR Heteroduplex Analysis Permits Differentiation of Transgene and Host Gene Expression in a Transgenic Animal Model

RT-PCR Heteroduplex Analysis Permits Differentiation of Transgene and Host Gene Expression in a Transgenic Animal Model

Blog Article

In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene.The potential for gene silencing may complicate matters further.Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences.We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from bovi-shield gold fp 5 l5 both human and mouse mRNA samples.Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls click here were tested.

Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples.In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling.In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time.The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design speciesspecific RT-PCR primers.