Huss David, Lansford Rusty
Developmental Neuroscience Program, Department of Radiology, Saban Research Institute, Children's Hospital Los Angeles, 4661 Sunset Boulevard MS #135, Los Angeles, CA, 90027, USA.
Translational Imaging Center, University of Southern California, Los Angeles, CA, 90089, USA.
Methods Mol Biol. 2017;1650:125-147. doi: 10.1007/978-1-4939-7216-6_8.
Real-time four-dimensional (4D, xyzt) imaging of cultured avian embryos is an ideal method for investigating the complex movements of cells and tissues during early morphogenesis. While methods that transiently label cells, such as electroporation, are highly useful for dynamic imaging, they can also be limiting due to the number and type of cells that can be effectively targeted. In contrast, the heritable, stable, and long-term expression of a fluorescent protein driven by the exogenous promoter of a transgene overcomes these challenges. We have used lentiviral vectors to produce several novel transgenic quail lines that express fluorescent proteins either ubiquitously or in a cell-specific manner. These lines have proven to be useful models for dynamic imaging and analysis. Here, we provide detailed protocols for generating transgenic quail with the emphasis on producing high titer lentivirus , effectively introducing it into the early embryo and efficiently screening for G1 founder birds .
对培养的禽类胚胎进行实时四维(4D,xyzt)成像,是研究早期形态发生过程中细胞和组织复杂运动的理想方法。虽然诸如电穿孔等瞬时标记细胞的方法对动态成像非常有用,但由于能够有效靶向的细胞数量和类型有限,它们也存在局限性。相比之下,由转基因的外源启动子驱动的荧光蛋白的可遗传、稳定和长期表达克服了这些挑战。我们已经使用慢病毒载体产生了几种新型转基因鹌鹑品系,这些品系以普遍表达或细胞特异性方式表达荧光蛋白。这些品系已被证明是动态成像和分析的有用模型。在这里,我们提供了生成转基因鹌鹑的详细方案,重点是生产高滴度慢病毒、有效地将其引入早期胚胎以及高效筛选G1代奠基鸟。