Knock-in mice and MEFs expressing active site mutant <i>Rev3l</i> have knockout phenotypes.
2016-01-05T14:56:08Z (GMT) by
<p>(A) Diagram of the mouse <i>Rev3l</i> ASM knock-in allele, with the wild-type (WT) locus shown at the top. Green rectangles indicate <i>Rev3l</i> coding sequences and the gray line represents chromosomal sequence. In the middle diagram, FRT sites are represented by double red triangles, loxP sites by blue triangles and lox511 sites by yellow triangles. The targeted exon 27 (starred) carries D2773A and D2775A point mutations and is inserted in an inverted orientation between wild-type exons 26 and 27. Splicing donor and acceptor sites flanking wild-type and ASM exon 27 are kept intact. The knock-in was produced by a Cre-dependent genetic switch. First, the neomycin positive selection cassette (neo) was excised by breeding with C57BL/6 Flp deleter mice. A subsequent cross with Cre-expressing mice led to excision of the wild-type exon 27 and inversion of ASM mutant exon 27 into the functional orientation. In the constitutive ASM knock-in locus shown in the lower diagram, the D2773A/D2775A <i>Rev3l</i> gene is expressed under the control of the endogenous <i>Rev3l</i> promoter and wild-type <i>Rev3l</i> exon 27 is absent from the locus. Heterozygous ASM knock-in mice (<i>Rev3L</i><sup>+/M</sup>) were then used for breeding. (B) Example of Southern blot analysis of (left) the inducible knock-in locus (<i>neo</i><sup>+</sup>) and (right) the constitutive ASM knock-in locus. Genomic DNA of the tested animals was compared with C57BL/6 wild-type genomic DNA (WT). <i>Eco</i>RV digested DNA was blotted on a nylon membrane and hybridized with the external 3’ probe with the position shown at the top of part A. Restriction fragments of 15 kb, 11.5 kb and 9.5 kb were observed for the wild-type, inducible knock-in locus (<i>neo</i><sup>+</sup>) and constitutive ASM knock-in locus, respectively. Genomic DNA was further analyzed extensively and confirmed by specific PCR assays and complete DNA sequencing as described in the Materials and Methods. (C) Genotypes of mouse pups produced by breeding parental <i>Rev3l</i><sup>+/M</sup> mice. (D) Growth of <i>Rev3l</i><sup>+/Δ</sup> and <i>Rev3l</i><sup>M/Δ</sup> cells. These cells were produced by addition of AdCre to <i>Rev3l</i><sup>M/lox</sup> or <i>Rev3l</i><sup>+/lox</sup> MEFs, deleting the floxed allele of <i>Rev3l</i>. (E) Survival of <i>Rev3l</i><sup>+/lox</sup>, <i>Rev3l</i><sup>M/lox</sup>, <i>Rev3l</i><sup>+/Δ</sup> and <i>Rev3l</i><sup>M/Δ</sup> primary MEFs 120 hr after addition of cisplatin, as measured by ATP content. (F) The MEFs as in (E) were stained with DAPI, and for 53BP1 and γ-H2AX by immunofluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005759#pgen.1005759.g002" target="_blank">Fig 2</a> to detect foci of DNA double-strand breaks. The quantification shows the percentage of cells with >2 53BP1 and γ-H2AX foci in <i>Rev3l</i><sup>+/lox</sup>, <i>Rev3l</i><sup>M/lox</sup>, <i>Rev3l</i><sup>+/Δ</sup> and <i>Rev3l</i><sup>M/Δ</sup> primary MEFs 9 days after AdCre treatment. (*) p < 0.01. Data represent mean ± SEM.</p>