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

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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 natio celebrate eyeshadow palette binding domain of the p53 gene from both human and mouse mRNA samples.Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls 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 here 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.