RT Journal Article SR Electronic T1 Rapid and precise genotyping of transgene zygosity in mice using an allele-specific method JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202201729 DO 10.26508/lsa.202201729 VO 6 IS 6 A1 Yang, Jianqi A1 DeVore, Alison N A1 Fu, Daniel A A1 Spicer, Mackenzie M A1 Guo, Mengcheng A1 Thompson, Samantha G A1 Ahlers-Dannen, Katelin E A1 Polato, Federica A1 Nussenzweig, Andre A1 Fisher, Rory A YR 2023 UL https://www.life-science-alliance.org/content/6/6/e202201729.abstract AB 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 (Atm TgC3001L, Nes-Cre, and Syn1-Cre) 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.