Cooper Caitlin A, Challagulla Arjun, Jenkins Kristie A, Wise Terry G, O'Neil Terri E, Morris Kirsten R, Tizard Mark L, Doran Timothy J
CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, VIC, Australia.
Transgenic Res. 2017 Jun;26(3):331-347. doi: 10.1007/s11248-016-0003-0. Epub 2016 Nov 28.
Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.
生成转基因和基因编辑哺乳动物涉及对卵母细胞或单细胞胚胎进行体外操作。由于禽类卵母细胞和单细胞胚胎相对难以获取，因此已经开发出了新的方案来生产转基因和基因编辑鸟类。虽然这些方案相对高效，但在获得完全体细胞和生殖系表达的转基因或基因编辑鸟类之前，需要经过两个世代间隔。这项工作大多是在鸡身上完成的，许多方案需要对原始生殖细胞（PGC）进行体外培养。然而，对于许多其他鸟类物种来说，不存在PGC长期培养的方法。开发第一代就能产生生殖系转基因或基因编辑鸟类的方法将节省大量时间和资源。此外，开发能够容易地应用于多种鸟类物种的方案将开辟新的研究机会。在此，我们报告一种使用精子作为基因编辑载体递送机制的方法，我们称之为精子转染辅助基因编辑（STAGE）。我们已成功使用该方法生成绿色荧光蛋白（GFP）基因敲除胚胎和鸡，以及使用CRISPR/Cas9系统生成在双性和mab-3相关转录因子1（DMRT1）基因中具有突变的胚胎。该方法的效率从低至0%到高至26%不等，诸如CRISPR引导效率和信使核糖核酸（mRNA）稳定性等多种因素可能会影响结果。这种简单直接的方法可以简化许多鸟类物种的基因编辑，包括那些目前尚无相关方法的物种。
