Poynter Greg, Huss David, Lansford Rusty
Division of Biology and Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA.
Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.emo112. doi: 10.1101/pdb.emo112.
The ability to generate transgenic mice has been a powerful tool in studying functional genomics, and much of our knowledge about developmental biology has come from the study of chicken embryology. Unfortunately, the availability of molecular genetic techniques, such as transgenics and knockouts, has been limited for developmental biologists using avian animal models. Efforts to develop a system for the rapid production of transgenic chickens have met with many obstacles, including high animal husbandry costs and long generational times. Recently, the Japanese quail has proven to be an excellent model organism for the production of transgenic avians using lentiviral vectors. The relatively small size of the adults, short time to sexual maturity, and prodigious egg production of the Japanese quail make development of transgenic lines less labor- and space-intensive compared to chickens. The high degree of homology between chicken and quail genomes allows researchers to design highly specific DNA constructs for the production of transgenic birds. In addition, transgenic quail offer all of the advantages of the classic avian developmental model system, such as the ability to readily produce quail:chick transplant chimeras. Finally, Japanese quail are ideal for in ovo imaging of embryos expressing fluorescent reporters introduced from a transgene and/or electroporation. Here, we provide detailed methods for generating transgenic quail using high-titer lentivirus.
生成转基因小鼠的能力一直是研究功能基因组学的有力工具，我们对发育生物学的许多认识都来自于对鸡胚胎学的研究。不幸的是，对于使用禽类动物模型的发育生物学家来说，诸如转基因和基因敲除等分子遗传技术的可用性有限。开发一种快速生产转基因鸡的系统的努力遇到了许多障碍，包括高昂的畜牧成本和较长的世代时间。最近，事实证明日本鹌鹑是使用慢病毒载体生产转基因禽类的优秀模式生物。与鸡相比，成年日本鹌鹑体型相对较小、性成熟时间短且产蛋量大，这使得转基因品系的开发在劳动力和空间方面的投入较少。鸡和鹌鹑基因组之间的高度同源性使研究人员能够设计出高度特异性的DNA构建体来生产转基因鸟类。此外，转基因鹌鹑具有经典禽类发育模型系统的所有优点，例如能够轻松产生鹌鹑：鸡移植嵌合体。最后，日本鹌鹑非常适合对通过转基因和/或电穿孔引入的荧光报告基因表达的胚胎进行胚胎内成像。在这里，我们提供了使用高滴度慢病毒生成转基因鹌鹑的详细方法。
