Jessen J R, Meng A, McFarlane R J, Paw B H, Zon L I, Smith G R, Lin S
Institute of Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.
Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5121-6. doi: 10.1073/pnas.95.9.5121.
The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.
通过同源重组对酵母人工染色体进行修饰已成为研究基因功能和增强子/启动子活性的一种有用的遗传工具。然而，要纯化出浓度足以满足多种应用的完整酵母人工染色体DNA却很困难。细菌人工染色体（BAC）是能够容纳大片段DNA的载体，并且能够像质粒DNA一样轻松纯化。我们在此报告一种简单的方法，即使用含有恰当定位的Chi位点的靶向构建体，通过同源重组对BAC进行修饰。为了证明该技术的用途，我们通过用绿色荧光蛋白（GFP）报告基因替换第一个编码外显子，对包含斑马鱼GATA - 2基因组位点的BAC克隆进行了修饰。分子分析证实，修饰过程中BAC没有额外的缺失或重排。显微注射表明，通过使用这些GFP修饰的GATA - 2 BAC，可以在活的斑马鱼胚胎中重现GATA - 2的表达模式。与较小的质粒构建体相比，显微注射修饰后的BAC克隆的胚胎嵌合性更低，造血祖细胞中的GFP表达也有所改善。通过Chi刺激的同源重组对BAC进行精确修饰，对于在基因转移适用的培养细胞或生物体中研究基因功能和调控应该是有用的。
