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Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method
Journal article   Open access   Peer reviewed

Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method

Jianqi Yang, Alison N DeVore, Daniel A Fu, Mackenzie M Spicer, Mengcheng Guo, Samantha G Thompson, Katelin E Ahlers-Dannen, Federica Polato, Andre Nussenzweig and Rory A Fisher
Life science alliance, Vol.6(6), e202201729
06/2023
DOI: 10.26508/lsa.202201729
PMCID: PMC10087101
PMID: 37037594
url
https://doi.org/10.26508/lsa.202201729View
Published (Version of record) Open Access

Abstract

Precise determination of transgene zygosity is essential for use of transgenic mice in research. Because integration loci of transgenes are usually unknown due to their random insertion, assessment of transgene zygosity remains a challenge. Current zygosity genotyping methods (progeny testing, qPCR, and NGS-computational biology analysis) are time consuming, prone to error or technically challenging. Here, we developed a novel method to determine transgene zygosity requiring no knowledge of transgene insertion loci. This method applies allele-specific restriction enzyme digestion of PCR products (RE/PCR) to rapidly and reliably quantify transgene zygosity. We demonstrate the applicability of this method to three transgenic strains of mice ( Tg , , and ) harboring a unique restriction enzyme site on either the transgene or its homologous sequence in the mouse genome. This method is as accurate as the gold standard of progeny testing but requires 2 d instead of a month or more. It is also exceedingly more accurate than the most commonly used approach of qPCR quantification. Our novel method represents a significant technical advance in determining transgene zygosities in mice.
 Alleles Animals Base Sequence Genotype Mice Mice, Transgenic Transgenes - genetics

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