PMC

Development of mrad50 mutant embryos. (a–f) Hematoxylin/eosin staining of sagittal sections of wild-type embryos (a, c, and e) and mutant embryos (b, d, and f) at E6.0 (a and b), E6.5 (c and d), and E7.5 (e and f). Note the smaller size of the mutant embryos. By E6.5, the homozygous mutant embryo has not developed appreciably beyond E6.0 stage. In many cases, the mutant embryo is in the process of being resorbed. By E7.5, the homozygous mutant embryo is completely resorbed. (Bar = 100 μm.) (g–j) BrdUrd labeling of proliferating cells in wild-type (g and i) and mutant (h and j) embryos at E6.0 and E6.5, respectively.

Creation of nullizygous cells in the presence of pmRad50 expression plasmid. (a) Southern blot of NcoI-digested DNA from m2/c1 cells before (lane 1) and after (lane 2) Cre-mediated recombination event deleting exons 1 and 2 in the presence of pmRad50. External probe 5P hybridizes to a 13-kb m2-specific fragment and a 7.8-kb c1-specific fragment. (b) mRad50 expression in cDNA-complemented m2/m2 cell lines. Fractionated extracts of wild-type (lanes 1–3) or m2/m2 complemented with pmRad50 (lane 4) cells, immunoblotted with mRad50 mAb. Wild-type whole-cell extract (lane 1); wild-type cell extracts immunoprecipitated with preimmune serum (lane 2) or mMre11 antiserum (lane 3); and m2/m2/pmRad50 cell extract immunoprecipitated with mMre11 antiserum (lane 4).

In vitro culture of blastocysts. (a and c) Blastocyst explants after 6 days without radiation treatment. Proliferation of both the inner cell mass (ICM) and the trophoblast giant cells (TGC) is prominent in both the wild-type (a) and mutant (c) embryos. (b and d) Blastocyst explants 6 days after 2 Gy γ-irradiation. Notice the active proliferation of both the inner cell mass and the trophoblast cells in the wild-type embryo (b). In contrast, in the mutant embryo, the inner cell mass is completely ablated (d). (e) Typical PCR genotyping of the blastocyst explants at the end of the experiment.

Gene targeting at the mRad50 locus. (a) Structure of the mRad50 locus and targeting vectors pTV1 and pTV2. The Southern blot hybridization analysis strategy was the same for the identification of targeted events generated by both targeting vectors. (b) Targeted clones first were identified by using a 5′ external probe (5P), which detects the change of a 15-kb wild-type NcoI fragment to a novel 7.8-kb NcoI fragment, and then were confirmed with a 3′ external probe (3P), which detects the change of a 17-kb wild-type EcoRV fragment into a novel 14-kb EcoRV fragment. The targeted allele generated by pTV1 is a null allele (designated mrad50^Brdm1 or m1). The targeted allele using pTV2 as a vector is a conditional null allele (mrad50^Brdc1 or c1). Cre recombinase-mediated removal of the PGK-neo/HSVtk cassette plus exons 1 and 2 of the mRad50 allele results in a null allele (the mrad50^Brdm2 or m2 allele), changing the 7.8-kb mrad50^Brdc1 allele NcoI fragment into a 13-kb NcoI fragment. The primers indicated by arrows enable PCR identification of the wild-type (5Rad50t1 + 3Rad50t1) and mrad50^Brdm1 alleles (mRad50t1 + 3Rad50t1). (c) Identification of a targeted clone from the pTV2 targeting vector. A targeted event is indicated by an arrow. The 15-kb NcoI fragment of the wild-type (wt) allele and the 7.8-kb fragment of the primary targeted (c1) allele are indicated. (d) Removal of the PGK-neo/HSVtk cassette plus exons 1 and 2 of mRad50 from the c1 allele by Cre to generate the m2 allele. The parental cell line is indicated by an arrow. The 13-kb NcoI fragment of the m2 allele is also indicated. (e) Gene targeting in mRad50/mrad50^Brdm2 ES cells with pTV1. Two clones in which the m2 mutant allele was retargeted to generate a null allele, mrad50^Brdm1 (m1), are indicated (arrows). (f) Gene targeting with pTV1 in trisomic chromosome 11 mutant ES cells that contain two copies of the wt allele and one copy of the m2 allele. Retargeting of the mutant allele is indicated by an arrow whereas targeting of one of two wt alleles is indicated by an open arrowhead.