McClelland Kathryn S, Wainwright Elanor N, Bowles Josephine, Koopman Peter
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
PLoS One. 2015 Jan 28;10(1):e0114932. doi: 10.1371/journal.pone.0114932. eCollection 2015.
Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.
传统的小鼠基因靶向方法复杂且耗时，尤其是在需要条件性缺失方法时。在此，我们描述了一种新技术，通过将修饰的反义吗啉代寡核苷酸（MOs）注射到妊娠中期小鼠胚胎的心脏中来评估基因功能。在让MOs通过胚胎血管循环后，将靶组织取出、培养并分析关键标志物的表达。我们通过部分模拟胎儿性腺（Stra8、Sox9）和胰腺（Sox9）中已知基因敲除表型建立了原理验证。我们还生成了Gli1和Gli2的新型双敲低，揭示了胎儿睾丸中Leydig细胞分化的缺陷。最后，我们深入了解了Adamts19和Ctrb1在性别决定和性腺发育中功能未知的基因的作用。这些研究揭示了该方法作为在进行详细遗传分析之前对器官发生过程中基因功能进行首过分析手段的实用性。
