Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells.
Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells.
复制标题
将完整的 200 至 500 kb YAC DNA 片段显微注射到哺乳动物细胞中。
作者：
A. Gnirke;C. Huxley;K. Peterson;M. Olson
酵母人工染色体（YACs）的DNA制备显微注射从大多数的天然酵母染色体上的脉冲场凝胶，琼脂糖酶处理，离心分离。100 mM NaCl的盐浓度是必要的，以保护DNA在这些程序中免受剪切。将590-kb的YAC，yGART 2，注射到中国仓鼠卵巢细胞中，产生表达YAC上携带的40-kb人GART基因的细胞。分析的12个细胞系中有9个含有围绕GART基因的至少110 kb的完整YAC DNA片段，一个细胞系含有至少480 kb，但不是完整的590 kb。小鼠LA-9细胞类似地注射含有人β-珠蛋白基因簇和哺乳动物选择标记的230-kb YAC的DNA。10个细胞系中有7个含有两个YAC载体臂加上完整的140 kb SfiI片段，跨越β-珠蛋白基因。通过RecA辅助限制性内切酶（RARE）切割分析了三个细胞系，发现含有完整的210 kb YAC插入片段。通过脂质体转染将类似制备的DNA引入哺乳动物细胞中产生具有多个YAC片段的细胞系，所述多个YAC片段通常比显微注射细胞系中发现的YAC片段短。结果表明，将凝胶纯化的YAC DNA显微注射到哺乳动物细胞中是一种有效的方法，可以将数百个DNA片段转移到哺乳动物细胞中。
DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.