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.Transcription factor GATA-2 is exemplary of a vital developmental factor whose regulation reveals many of the intricacies of the controlling apparatus that is required for proper temporal and tissue-specific expression of a gene in many different tissues and organs that are critical for mammalian development. The evolutionarily conserved C 4 zinc finger GATA transcription factors play demonstrably crucial roles in embryogenesis. GATA-2, originally cloned from a chicken cDNA library (1), was shown to be essential for the proliferation and/or differentiation of early hematopoietic progenitors, as Gata2 null mutant mice die around mid-gestation from a block in primitive hematopoiesis (2). Further examination of Gata2 gain-of-function mice and in vitro differentiation of Gata2 null mutant ES cells underscored the fundamental conclusions from the initial loss of function experiments, and showed that GATA-2 plays a pivotal role in the proliferation of very early hematopoietic progenitors (3-5).We showed several years ago that Gata2 null mutant embryonic lethality could be rescued by complementation with a 247-kbp transgenic yeast artificial chromosome (YAC) 3 bearing sequences from Ϫ174 to ϩ73 kbp (revised endpoints relative to the Gata2 translational initiation site); the YAC contains all of the regulatory information required to rescue Gata2 function in p...

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…Dissociation curves were obtained to confirm the specificity of the amplified DNA. The primer sequences were 5'-AGCAAAGACCCCAACGAGAA-3' and 5'-GGCGGCGGTCACGAA-3' for EGFP (Harraghy et al, 2011), and 5'-CCATAGGCTTCACACCTTCCTG-3' and 5'-GCACTAACACTACCTTCCTCAACCG-3' for -actin as an endogenous control of two copies (Brandt et al, 2008). A standard curve for each gene was generated using serial dilutions of genomic DNA and we confirmed that amplification efficiencies of both primer pairs were similar (data not shown).…”

“…We purified the genome DNA from the tail of each line of mouse and performed qPCR using primers to amplify the EGFP gene. We also amplified the endogenous -actin gene as a control of two copies (Brandt et al, 2008) and the copy number of the transgene was calculated as a ratio to the control per copy. Ratios of EGFP to -actin per copy were 2.96 ± 0.80 for #528, 2.60 ± 0.56 for #531, 0.87 ± 0.30 for #542, 1.37 ± 0.55 for #566, 1.16 ± 0.39 for #675, and 1.38 ± 0.27 for #676 (Table1).…”

Conditional Gata2 inactivation results in HSC loss and lymphatic mispatterning

“…More than a decade ago, we discovered that a 271-kbp Gata2 yeast artificial chromosome (YAC) transgene, which circumscribed genomic sequences from -198 kbp to +73 kbp (with respect to the translational start site) of the Gata2 locus, could fully rescue the embryonic lethality in Gata2 -/-embryos (3). Hence, this YAC functionally defined the genomic limits of the Gata2 locus that contained all of the regulatory element(s) required for fully elaborating primitive and definitive hematopoiesis, but the YAC only incompletely rescued all of the GATA-2 functions, since the P0 pups died from incomplete patterning of the urogenital system (3,9,20).…”

A mouse model of adult-onset anaemia due to erythropoietin deficiency

“…Mutation of the start codon was also introduced in the first exon to initiate translation from the start codon of Cre cDNA. The recombinant BAC clone was confirmed by digestion and sequencing, and purified as circular BAC DNA by ultra centrifugation with caesium chloride 55 .…”