Claire J. Houchen, Maria Bergman-Gonzalez, Erin E. Bumann
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引用次数: 0
摘要
性别是所有生物医学实验中必须考虑的一个生物变量。然而,要在鸟类胚胎中考虑性别问题却很有难度,因为性别在形态上是无法区分的。与人类不同,雌性鸟类是具有 Z 和 W 性染色体的异性性别。以前曾利用物种特异性聚合酶链反应(PCR)技术在雏鸟体内鉴定出雌鸟特异性 W 染色体。我们开发了一种新型反转录定量 PCR(RT-qPCR)技术,可扩增雏鸡、鹌鹑和鸭的 W 染色体基因组氨酸三核苷酸结合蛋白 W(HINTW)。通过使用物种特异性 PCR(包括新型鹌鹑特异性 HINTW PCR 引物集),在所有三个物种中证实了 HINTW RT-qPCR 引物集的准确性。然后按性别分析了鸭和鹌鹑胚胎下颚中与骨骼发育相关的基因表达,因为已知成年鸭的喙大小具有性别二态性,而鹌鹑的喙大小则不具有性别二态性。结果发现,鸭的基因表达有性别差异的趋势,而鹌鹑则没有,这是意料之中的。有了这些新的 RT-qPCR 和 PCR 胚胎性别鉴定方法,现在就可以通过 RNA 和 DNA 来评估雏鸡、鹌鹑和鸭胚胎的性别,这有助于在使用这些模式生物进行研究时将性别作为一个生物变量进行分析。
A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone
Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.