Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.
Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.
Xiao
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Department of Microbiology and Molecular Genetics, Harvard Medical School and Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
Weaver
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D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D004260:DNA Repair; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018390:Gene Targeting; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D029701:Saccharomyces cerevisiae Proteins; D013234:Stem Cells; D014764:Viral Proteins
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae