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.

基因组编辑工具的进步减少了创建动物模型的障碍。由于它们在解剖学和生理学上与人类相似，因此越来越需要猪模型来研究人类疾病、异种移植和转化研究。确定转基因胚胎、细胞或胎儿性别的能力对每个涉及转基因动物生产的项目都有好处。这种策略可以提高时间效率并降低生产成本。此外，性别评估对于野生动物研究非常有用，可以了解种群行为和结构。因此，我们通过使用独特的引物组来扩增DDX3X或DDX3Y，开发了一种简单快速的基于 PCR 的方案，用于猪的性别确定基因。当使用来自猪的尾部基因组 DNA、第 35 天胎儿和毛发样本时，性别被 100% 正确分配。对于囊胚和卵母细胞（分别为 84.6% 和 96.5% 的功效），未鉴定的样本可能是由于样本量的限制。我们的分析也适用于家养绵羊 ( Ovis aries )、美洲野牛 ( Bison bison ) 和欧洲牛 ( Bos taurus ) 样本，通过计算机分析，我们确认了其他 12 种哺乳动物中 DDX3 基因的 X-Y 扩增子长度多态性。这种用于确定猪组织和细胞性别的 PCR 方案显示出简单、特异、高度可重复且耗时少，是其他牲畜物种和野生动物研究的重要工具。