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.
Highlights
Transcription factor GATA-2 is exemplary of a vital developmental factor whose regulation reveals many of the intrica
Because the rescuing yeast artificial chromosome (YAC) was not expressed in the urogenital system, while endogenous GATA-2 is prominently expressed there, we speculated that the development of hydroureters and cystic kidneys was due to the absence of tissue-specific enhancers that were responsible for directing proper Gata2-mediated patterning of the developing urogenital system in the YAC transgene
Expire from urogenital patterning failures (6), it seemed reasonable to ask: if both of these embryonic deficiencies were complemented by a transgene bearing activities that would correct both deficiencies, would the animals survive? The alternative, which might have been expected in this case, is that the animals would probably not survive to reproductive age, but expire of a second perinatal lethal urogenital mented Gata2Ϫ/Ϫ:TgG2BAC compound mutant animals as in failure (due to the anticipated absence from the transgene of heterozygous mutant (Gata2ϩ/Ϫ) controls (Fig. 4, B and F). any urogenital epithelia-complementing activity), or even from
Summary
BACs—BACs spanning the mouse Gata locus were identified (16), purified (17), and modified (7) as previously described. The neomycin gene was again removed by cre induction, leaving a single loxP514 site at the Ϫ13 kbp position This BAC was further modified to insert an Frt-flanked ampicillin resistance gene to allow for selection of products that had undergone intermolecular recombination. This was achieved by using Famp2977 and Famp4233 primers (supplemental Table S1) together with the FrtAmp plasmid to generate a targeting fragment that was inserted into 81F7lox BAC by homologous recombination, and in doing so, inserted the FrtAmp cassette between positions 2977 and 4233 of the pBACe3.6 vector backbone (21). Linked BAC Transgenic Mice— Due to fragmentation problems encountered when very large linear
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