Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis

Abstract

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.

Figure 1

Schematic representation of the zebrafish GATA-2 genomic locus before and after modification by the Chi-containing G2TC DNA fragment. (A) Genomic structure of the three GATA-2 BAC clones and their relationships to the ATG initiation codon. Arrows indicate the orientation of the GATA-2 gene. (B) Region of the wild-type GATA-2 gene locus targeted for homologous recombination. (C) The nonreplicative G2TC DNA fragment containing Chi sites properly situated to promote homologous recombination (solid arrows). The edges of the 5′ and 3′ regions of homology with the GATA-2 gene locus are indicated by dashed lines. (D) GATA-2 gene locus after replacement of the first coding exon with the GFP and kanamycin genes. This modification adds several restriction sites to the GATA-2 gene as follows: B, BamHI; C, ClaI; Rv, EcoRV; H, HindIII; N, NotI; Sc, SacI; S, SalI; X, XhoI. Primers P1/P2 and P3/P4 were used in a PCR to identify BAC DNA with the correct gene replacement. Primers P1 and P4 were used to sequence the edges of the 5′ and 3′ regions of homology, respectively.

Figure 2

Southern blot analysis of three different GATA-2 BAC clones modified by the G2TC DNA fragment. Newly introduced restriction sites (Fig. 1D) were used to digest both wild-type (lanes 1) and modified (lanes 2) BAC DNA. (A) GATA-2 BAC clone 3 was digested with the indicated enzymes and probed with a 2-kb wild-type ClaI–EcoRV genomic fragment (Fig. 1B). A 9-kb wild-type EcoRV fragment became 8.8-kb and 2-kb fragments. An 11-kb wild-type XhoI–BamHI fragment became 9-kb and 2-kb fragments. A 20-kb wild-type SalI fragment became 12-kb and 8.6-kb fragments. A 10.5-kb wild-type ClaI fragment became 9.5-kb and 1.7-kb fragments. A 13.5-kb wild-type HindIII fragment became 12-kb and 2.5-kb fragments. GATA-2 BAC clones 1 and 4 (B) also contained the desired gene replacement.

Figure 3

GFP expression from modified GATA-2 BACs in living zebrafish embryos. (A) GFP expression driven by modified BAC clone 4 in the ventral ectoderm and mesoderm of a 6-h shield stage embryo. Arrow indicates the dorsal shield. (B) GFP expression driven by modified BAC clone 4 in the posterior intermediate cell mass of a 20-h embryo. (C) GATA-2 expression pattern in an 18-h embryo as detected by RNA in situ hybridization. (D) GFP expression driven by modified BAC clone 3 in circulating hematopoietic cells of a 48-h embryo. GFP expression driven by modified BAC clone 4 in the EVL (E and G) and in a motoneuron (F) of 48-h embryos.