RT-PCR heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal model.
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 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 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 species-specific RT-PCR primers.
The Ohio State University College of Medicine and Public Health, Columbus 43210-1240, USA.
W
W
Duan
H
H
Ding
W G
WG
Zhu
K
K
Srinivasan
G A
GA
Otterson
M A
MA
Villalona-Calero
G A
GA
Ottersom
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001483:Base Sequence; D015870:Gene Expression / Q000235:genetics*; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D051379:Mice; D023421:Models, Animal; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment / Q000379:methods; D019076:Transgenes / Q000235:genetics*