• Jason R. Jessen
    Institute of Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912
  • Anming Meng
    Institute of Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912
  • Ramsay Mcfarlane
  • Barry H. Paw
    Harvard Medical School
  • Leonard I. Zon
    Harvard Medical School
  • Gerald R. Smith
    Fred Hutchinson Cancer Research Center, Seattle,
  • Shuo Lin
    Institute of Molecular Medicine and Genetics and Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912
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.
Original languageEnglish
Pages (from-to)5121
Number of pages5126
JournalProceedings of the National Academy of Sciences of the USA
Volume95
Issue number9
Publication statusPublished - 1998
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