Rani Pyapalli Usha, Khillan Jaspal Singh
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Transgenic Res. 2003 Dec;12(6):739-41. doi: 10.1023/b:trag.0000005248.50923.cf.
Gene targeting in embryonic stem (ES) cells followed by preparation of chimeric animals is the most effective method to study the function of a gene during development and differentiation. Here, we describe a cost effective and convenient method to produce chimeric animals. Cryopreserved 8-16 cell mouse embryos were aggregated with ES cells in microwell petridishes (Khillan & Bao, 1997) to obtain blastocysts. Also, freshly isolated morulas were aggregated with ES cells that were positive for the green florescent protein (GFP). After overnight culture, the blastocysts that exhibited GFP florescence were transferred to the pseudo-pregnant mothers to obtain chimeric animals. The animals displayed high degree of ES contribution and transmitted gene to their progeny after mating with the normal animals. The studies demonstrate that the aggregation with cryopreserved embryos followed by the pre-selection for a visual marker can be a high throughput and cost effective method to create chimeric animals from the gene targeted ES cells.
在胚胎干细胞（ES细胞）中进行基因靶向，随后制备嵌合体动物，是研究基因在发育和分化过程中功能的最有效方法。在此，我们描述一种经济高效且便捷的制备嵌合体动物的方法。将冷冻保存的8 - 16细胞期小鼠胚胎与ES细胞在微孔培养皿中聚合（Khillan和Bao，1997）以获得囊胚。此外，将新鲜分离的桑葚胚与绿色荧光蛋白（GFP）呈阳性的ES细胞聚合。过夜培养后，将显示GFP荧光的囊胚转移至假孕母鼠体内以获得嵌合体动物。这些动物显示出高度的ES细胞贡献，并且在与正常动物交配后能将基因传递给它们的后代。研究表明，与冷冻保存的胚胎聚合，随后对视觉标记进行预选，可能是一种从基因靶向的ES细胞创建嵌合体动物的高通量且经济高效的方法。
