Gnirke A, Huxley C, Peterson K, Olson M V
Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110.
Genomics. 1993 Mar;15(3):659-67. doi: 10.1006/geno.1993.1121.
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.
酵母人工染色体（YACs）的DNA通过在脉冲场凝胶上与大多数天然酵母染色体分离、用琼脂酶处理并离心来制备用于显微注射。在这些操作过程中，100 mM NaCl的盐浓度对于保护DNA免受剪切是必要的。将一个590 kb的YAC，yGART2，注射到中国仓鼠卵巢细胞中，产生了表达YAC上携带的40 kb人类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。类似地，将含有人类β-珠蛋白基因簇和一个哺乳动物选择标记的230 kb YAC的DNA注射到小鼠L A-9细胞中。所得的10个细胞系中有7个含有YAC载体臂以及跨越β-珠蛋白基因的完整140 kb SfiI片段。通过RecA辅助的限制性内切酶（RARE）切割分析了3个细胞系，发现它们含有完整的210 kb YAC插入片段。通过脂质转染将类似制备的DNA导入哺乳动物细胞产生了具有多个YAC片段的细胞系，这些片段通常比显微注射细胞系中的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将几百千碱基对大小的DNA片段转移到哺乳动物细胞中的一种有效方法。
