Generation of mice harbouring a conditional loss-of-function allele of Gata6
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The zinc finger transcription factor GATA6 is believed to have important roles in the development of several organs including the liver, gastrointestinal tract and heart. However, analyses of the contribution of GATA6 toward organogenesis have been hampered because Gata6-/- mice fail to develop beyond gastrulation due to defects in extraembryonic endoderm function. We have therefore generated a mouse line harbouring a conditional loss-of-function allele of Gata6 using Cre/loxP technology.
LoxP elements were introduced into introns flanking exon 2 of the Gata6 gene by homologous recombination in ES cells. Mice containing this altered allele were bred to homozygosity and were found to be viable and fertile. To assess the functional integrity of the loxP sites and to confirm that we had generated a Gata6 loss-of-function allele, we bred Gata6 'floxed' mice to EIIa-Cre mice in which Cre is ubiquitously expressed, and to Villin-Cre mice that express Cre in the epithelial cells of the intestine. We conclude that we have generated a line of mice in which GATA6 activity can be ablated in a cell type specific manner by expression of Cre recombinase. This line of mice can be used to establish the role of GATA6 in regulating embryonic development and various aspects of mammalian physiology.
KeywordsloxP Site Gata6 Gene Gata6 Locus Gata6 mRNA Decidual Mass
The mouse Gata6 gene encodes a 45 kD protein containing two highly conserved zinc-finger DNA binding domains with a Cys-X2-Cys-X17-Cys-X2-Cys motif that directs binding to the nucleotide sequence element (A/T)GATA(A/G) 1. GATA4, 5 and 6 make up a subset of GATA factors that have been implicated in the development of several organs including the heart, lung, gastrointestinal tract and liver [1, 2, 3, 4]. Development of GATA6 null embryos arrests during gastrulation as a consequence of defects in extraembryonic endoderm function [2, 5]. This early embryonic lethality can be rescued by complementing the GATA6 null embryos with a wild type extraembryonic visceral endoderm using tetraploid embryo complementation, and embryos derived by this process can survive until E10.5 . Analyses of such Gata6-/-ES cell-derived embryos has revealed defects in hepatogenesis, which supports the proposal that GATA6 is an important developmental regulator. Although the ability to generate embryos from Gata6-/-ES cells by tetraploid embryo complementation has provided important insight into the contribution of GATA6 during early embryogenesis, this approach is not compatible with studying the role of GATA6 at later stages in development or its role in controlling differentiation of specific cell types. In the current report, we describe the generation of mice containing a conditional null allele of Gata6 that can be used for cell type-specific removal of GATA6 by Cre-mediated recombination.
Results and discussion
Embryonic lethality associated with loss of GATA6 function
E8.5 – 11.5 embryos
E8.5 – 11.5 embryos
In summary, we conclude that we have generated a line of mice in which GATA6 transcriptional activity can be ablated by expression of Cre. We believe that the availability of this mouse will be useful to elucidate the contribution of GATA6 during organogenesis as well as its physiological role in adult mice.
A bacterial artificial chromosome (BAC # RP23-410I12) that contained Gata6 genomic DNA was obtained from BACPAC Resources, Children Hospital Research Institute, Oakland, CA. Plasmid pNeb-DT-GATA6 was generated by cloning a 12.2 kb EcoRV/PacI genomic fragment containing Gata6 exons 1 and 2 into the PacI/HincII site of a vector, pNEB193-DT, which contained a diphtheria toxin (DT) expression cassette to enrich against random integration of the targeting plasmid in ES cells. A cassette containing loxP-neo-loxP was amplified by PCR from the plasmid pSV-LNL (modified from Zhang et al) using the following primers: Gata6ET1: GCTTGCTGTTTGAGTCTACCCCATTTCTGCCTGTTTCTTGACATCCCTTCGAATTCTGGTACCGGCGCGCCTAGTCGAC, Gata6ET2: ATCCATTATTGTCAATGTCTAAAGATGGAATTGTCTCTGCACAAGCTATCTTCTCAACTCGAGCCCTTAATTAACCGGT. These oligonucleotides contained 55 bp of sequence from Gata6 intron 2 (underlined). This amplicon was introduced into Gata6 intron 2 sequence in pNEB-DT-GATA6 by homologous recombination in E.coli following the procedure described by Lee et al  The loxPneoloxP cassette was then converted to a single loxP site by expression of Cre recombinase . A loxP(FRTneoFRT) cassette was then amplified from pSV-LFNF using primers Gata6ET3: CACGCTGGTGGTTGTAAGGCGGTTTGTGTTTAAGGTGTGCGGTTGGCCTGGACGTGTGGTACCGGCGCGCCTAGTCGAC, Gata6ET4: GAAAAAGTTACCTAGCCCAGAGAAAGTGAGATGCCAGGAAAGGCATAAGGATATCAACTCGAGCCCTTAATTAACCGGT. These oligonucleotides contain 56 bp (Gata6ET3) and 52 bp (Gata6ET4) of sequence from Gata6 intron 1, respectively. This cassette was introduced into Gata6 intron 1, again using homologous recombination in E.coli. to generate the final targeting vector (Fig. 1A).
ES cell targeting and animals
Linear targeting vector (100μg) was introduced into R1 ES cells by electroporation, and the genotype of colonies resistant to 350μg/ml of Geneticin (Gibco BRL) was determined by Southern blot (Fig. 1B). Chimeric mice were generated by aggregation of ES cells with CD-1 morulae as described previously  and the modified allele was passed through the germline by breeding chimeras to CD1 mice. Gata6loxP/loxPmice were produced by breeding Gata6loxp(FRTneoFRT)/+ mice to B6;SJL-Tg(ACTFLPe)9205Dym/J mice  (Jackson Labs) to delete the FRTneoFRT cassette by Flp-mediated recombination in the germline. The ACTFLPe transgene was removed by breeding F1 Gata6loxP/loxPmice into CD-1 mice. Gata6+/delmice were generated by mating Gata6loxp(FRTneoFRT)/+ animals with B6.FVB-Tg(EIIa-cre)C5379Lmgd/J transgenic mice  (Jackson Labs) to allow Cre-mediate recombination between loxP elements in the germline. The EIIa-Cre transgene was removed by breeding Gata6+/delF1 mice with CD1 mice. The MCW IACUC committee approved all procedures using animals.
Southern blot, PCR and RT-PCR
Southern blot analyses were performed using standard conditions with probes indicated in Fig. 1. Genotypes were determined by PCR using the following oligonucleotide primer pairs: Gata6 gt4F/4R, GTGGTTGTAAGGCGGTTTGT, ACGCGAGCTCCAGAAAAAGT; Gata6 k/o2F/2R, AGTCTCCCTGTCATTCTTCCTGCTC, TGATCAAACCTGGGTCTACACTCCTA; Flp F/R, GGTCCAACTGCAGCCCAAGCTTCC, GTGGATCGATCCTACCCCTTGCG ; Cre 1/2, GTTCGCAAGAACCTGATGGACA, CTAGAGCCTGTTTTGCACGTTC ; Neo A/B, GCCAACGCTATGTCCTGATAGCGGT, AGCCGGTCTTGTCGATCAGGATGAT. RT-PCR was performed as described previously  with the following primer pairs: Gata6 9/10; AGTTTTCCGGCAGAGCAGTA, AGTCAAGGCCATCCACTGTC, Pol2 F/R; CTGATGCGGGTGCTGAGTGAGAAGG, GCGGTTGACCCCATGACGAGTG.
Immunohistochemistry was performed using antigen retrieval in citrate buffer as described previously  using an anti-GATA6 antibody (AF1700 R&D Systems,1/1000 dilution).
We would like to thank Drs. Robert Burgess and Francis Stewart for providing plasmids and Dr. Neal Copeland for providing bacterial strains used in recombineering. Pregnant mare serum gonadotrophin (PMSG) used in superovulation was obtained from Dr. A.F. Parlow at the National Hormone and Peptide Program (Torrance, CA). We are also grateful to Dr. Michele Battle for critically evaluating the manuscript and for providing plasmids. This work was supported by an NIH training grant from the MCW cardiovascular research centre to C.P.S and NIH grants to S.A.D.
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