Chen S, Evans G A
Molecular Genetics Laboratory, Center for Human Genome Research, The Salk Institute for Biological Sciences, San Diego, CA.
Methods Mol Biol. 1993;15:75-80. doi: 10.1385/0-89603-244-2:75.
The production of transgenic mice by the direct microinjection of DNA fragments into isolated mouse embryos is now a standard technique for molecular and developmental analysis (1). Traditionally, the initial screening of litters of mice for those carrying the transgene has been carried out by the preparation of DNA from pieces of excised tail obtained at 2-3 wk of age ("tail blot"). The presence of the transgene is determined through Southern blot analysis, a procedure that usually takes a minimum of 5 d. Two important criteria for successful screening are that the tail DNA is sufficiently purified from connective tissues and proteins to enable restriction enzyme digestion, and that the amount of DNA is large enough to allow the detection by hybridization of radioisotope-labeled probes recognizing the transgene or endogenous gene sequences.
通过将DNA片段直接显微注射到分离的小鼠胚胎中来生产转基因小鼠,现在是分子和发育分析的标准技术(1)。传统上,对携带转基因的小鼠幼崽进行初步筛选是通过从2-3周龄时切除的尾巴片段制备DNA(“尾巴印迹”)来进行的。通过Southern印迹分析来确定转基因的存在,该过程通常至少需要5天。成功筛选的两个重要标准是,尾巴DNA要从结缔组织和蛋白质中充分纯化,以便进行限制性酶切,并且DNA的量要足够大,以允许通过杂交检测识别转基因或内源基因序列的放射性同位素标记探针。