Defining the functional boundaries of the Gata2 locus by rescue with a linked bacterial artificial chromosome transgene

Abstract

Transcription factor GATA-2 is vital for both hematopoietic progenitor cell function and urogenital patterning. Transgenic mapping studies have shown that the hematopoietic and urogenital enhancers are located hundreds of kbp 5' and 3' to the Gata2 structural gene, and both are vital for embryonic development. Because the size of mammalian genes, including all of their associated regulatory elements, can exceed a megabase, transgenic complementation in mice has, in specific instances, proven to be a formidable hurdle. After incorporating the Gata2 structural gene as well as the distant hematopoietic and urogenital enhancers into a single, contiguous piece of DNA by fusing two bacterial artificial chromosomes (BACs) into one, we formally tested the hypothesis that the functional boundaries of this locus are contained within this contiguous genomic span. We show that two independent lines of transgenic mice bearing a multicopy 413-kbp-linked Gata2 BAC transgene (bearing sequences from -187 to +226 kbp of the locus) are able to fully rescue Gata2 null mutant embryonic lethality and that the rescued animals behave and reproduce normally. Surprisingly, the linked BAC confers expression in the ureteric epithelium, whereas sequences within any of the overlapping parental BACs and a yeast artificial chromosome that were originally tested do not, and thus these experiments also define a novel synthetic enhancer activity that has not been previously described. These genetic complementation studies define the required outer limits of the Gata2 locus and formally demonstrate that enhancers lying beyond those boundaries are not necessary for Gata2-regulated viability or fecundity.

FIGURE 1.

Cre-mediated generation of a 413 kbp linked BAC. A, diagram depicting Gata2 and other genes (black boxes) on mouse chromosome 6 as well as BACs and a YAC (d16) that have been examined in current and previous studies. B, a strategy was devised for recombining two overlapping parental BACs (highlighted in blue in panel A) so that the combination of urogenital and hematopoietic enhancers could be examined as a single transgene. The relative positions of the Gata2 structural gene (hatched box) and the enhancers known to be critical for hematopoietic (red ovals) or urogenital (purple ovals) development are indicated. Site-specific cre-mediated recombination was employed to fuse the two parental BACs, 115E9 (P1) and 81F7 (P2), by simultaneous intermolecular homologous recombination between loxP514 (yellow arrowheads) and loxP511 (green arrowheads) sites. The strategy was predicted to generate a 413-kbp BAC bearing contiguous sequence from –187 to +226 kbp surrounding the Gata2 gene (G2BAC, right). The neomycin-positive selection marker was subsequently removed by Flp-mediated recombination between Frt sites (blue arrowheads). C, pulsed-field gel electrophoresis of the two parental (P1 and P2) BACs as well as a recombinant-linked BAC (R). The BACs were digested with PI-SceI; molecular weight markers (M) are λ DNA concatemers. D, HindIII restriction digest DNA fingerprinting shows that all of the recombinants (R1–R7) contain all of the bands within both parental BACs (P1 and P2). Molecular weight markers (M) for panels D-G are a mixture of HindIII- or BstEII-digested λ DNA samples. E–G, Southern blot analysis of the parental (P1 and P2) and linked recombinant (R1–R4) BACs display the anticipated sizes. E, probe E, located at –174 kbp (see panel B), hybridizes to a 3.9-kbp HindIII fragment from P1 (that is not detected in P2) and all recombinant BACs. F, hybridization to probe F, located at –13 kbp, generates a 5-kbp band in P1 as well as a 10-kbp band in P2 and all of the recombinant BACs after digestion with PmeI+PvuII. G, probe G, located at approximately +200 kbp, hybridizes to a 7-kbp HindIII fragment from P2 and the recombinant BACs, while no hybridization is detected in P1 DNA.

FIGURE 2.

Gata2-linked BAC copy number as determined by Q-PCR. Tail DNA was recovered from animals in which the transgenes in two different established lines (called 2.2 and 2.12) were stably transmitted to offspring for more than four generations. PCR primers were developed using Primer Express, which would detect unique sequences approximately every 50 kbp along the length of the linked BAC, as well as at positions outside the boundaries of the BAC (as a diploid copy number control; supplemental Table S1). The normalization strategy for each genomic DNA sample involved first standardizing the quantified PCR product from Gata2-specific primer pairs to that of the β-actin gene, and then normalizing the values of transgenic mice to those of wild-type controls.

FIGURE 3.

BAC-complemented Gata2^–/– mice are born in a Mendelian distribution. A, Q-PCR was performed on genomic DNA samples prepared from the tails of sibling pups (S1–S10) from a presumptive Gata2^+/–:Tg^G2BAC × Gata2^+/– intercross (primer sequences for the EII-KI allele are shown in supplemental Table S1, Q-eGFP); β-actin Q-PCR was used as a diploid copy number control. The strategy was designed to detect the EII-KI (Gata2^–) allele in all GFP⁺ mice (supplemental Table S1), and therefore wild-type mice have a ratio (Gata2^–/β-actin) of 0, EII-KI heterozygotes (e.g. both parents) have a ratio of 1, and EII-KI homozygotes (Gata2 null mutant mice) have a ratio of 2. B and C, representative semi-quantitative PCR results depicting detection of the linked BAC transgene allele (B, using BAC5′jxn primers, supplemental Table S1) or the EII-KI (–) or wild-type (+) alleles (C, referred to as GFP or wt alleles, supplemental Table S1) from a presumptive Gata2^–/–:Tg^G2BAC X Gata2^+/+ intercross. Note that every sibling (S1–S9) from this mating carries a (–) germ line allele, whereas only seven of nine sibs harbor a BAC allele, proving that the genotype of the original transgenic parent (P2) is Gata2^–/–:Tg^G2BAC. Note that the + allele can be contributed either through the germ line or from a BAC-derived Gata2 gene.

FIGURE 4.

The G2BAC transgene recapitulates mesenchymal and epithelial Gata2 urogenital expression. Transverse cryosections of e14.5 Gata2^+/– (A–D) and Gata2^–/–:Tg^G2BAC (E–H) embryos were subjected to co-immunostaining using anti-GFP (B and F) and anti-GATA-2 (C and G) antibodies, prior to hematoxylin and eosin histological staining (A and E). B and F, GFP immunofluorescence reflects the endogenous GATA-2 expression pattern in the urogenital mesenchymal and epithelial cells of Gata2^+/– and Gata2^–/–: Tg^G2BAC embryos. C and G, GATA-2 protein was immunologically detected in the urogenital mesenchymal and epithelial compartments of the Gata2^+/– embryo (C). In the Gata2^–/–:Tg^G2BAC embryo, anti-GATA-2 immunoreactivity was present not only in the urogenital mesenchyme, but also in the ureteric epithelium (G). D and H, merged images of anti-GFP and anti-GATA-2 immunofluorescence. e, ureteric epithelium; m, urogenital mesenchyme.