Abstract
Advances in genome editing tools have reduced barriers to the creation of animal models. Due to their anatomical and physiological similarities to humans, there has been a growing need for pig models to study human diseases, for xenotransplantation and translational research. The ability to determine the sex of genetically modified embryos, cells or fetuses is beneficial for every project involving the production of transgenic animals. This strategy can improve the time-efficiency and lower the production costs. Additionally, sex assessment is very useful for wildlife studies to understand population behavior and structure. Thus, we developed a simple and fast PCR-based protocol for sex determination in pigs by using a unique primer set to amplify either the DDX3X or DDX3Y gene. The sex was 100% correctly assigned when tail genomic DNA, Day-35 fetus and hair samples from pigs were used. For both blastocysts and oocytes (84.6% and 96.5% of efficacy, respectively) the unidentified samples were potentially due to a limitation in sample size. Our assay also worked for domestic sheep (Ovis aries), American bison (Bison bison) and European cattle (Bos taurus) samples and by in silico analysis we confirmed X–Y amplicon length polymorphisms for the DDX3 gene in 12 other mammalian species. This PCR protocol for determining sex in pig tissues and cells showed to be simple, specific, highly reproducible and less time consuming as well as an important tool for other livestock species and wildlife studies.
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Acknowledgements
We would like to thank Dr. Robert Schnabel, Msc. Emma Stephenson, MSc. Paula Chen, Msc. Eleonore O’neil and Dr. Jon Green for generously provide genomic DNA and tissue samples from different species to this present study. The results presented here were funded by the National Institutes of Health via funding from OD, NIAID and NHLBI via U42OD011140.
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The results presented here were funded by the National Institutes of Health via funding from OD, NIAID and NHLBI via U42OD011140.
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C. G. L., W. C. W, and K. D. W designed the experiments. C.G.L., A.M.S., M. S. S. and L. D. S. conducted the experiments. C.G.L., K. D. W., and R. S. P. analyzed the data and wrote the manuscript.
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Lucas, C.G., Spate, A.M., Samuel, M.S. et al. A novel swine sex-linked marker and its application across different mammalian species. Transgenic Res 29, 395–407 (2020). https://doi.org/10.1007/s11248-020-00204-z
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DOI: https://doi.org/10.1007/s11248-020-00204-z