Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice  Jie Zhu, Simone Petersen, Lino Tessarollo, André Nussenzweig  Current Biology  Volume 11, Issue 2, Pages 105-109 (January 2001) DOI: 10.1016/S0960-9822(01)00019-7

Figure 1 Targeting of mouse NBS1(a) Genomic structure of the wild-type NBS1 locus and targeting vector. A genomic clone was isolated from a mouse BAC library (Genome Systems) with a 700 bp BgII/PstI probe derived from the 5′ end of mouse NBS1 cDNA. In the targeting vector and targeted allele, the neomycin selection cassette replaces the first NBS1 exon, including the translation initiation codon. Homologous recombination introduces EcoRI (E) and HincII (H) sites, which reduces the 11 kb wild-type EcoRI fragment and the 3.0 kb wild-type HincII fragment to 2 kb. Four successful targeting events were identified in 250 clones screened. (b) Identification of NBS1 targeting in ES cells by southern blotting. EcoRI-digested genomic DNA from NBS1+/+ and NBS1+/− ES cells were hybridized with a 300 bp PstI probe upstream of exon 1; results were confirmed with HincII digestion of genomic DNA (not shown). (c) PCR screening of DNA from ES cells and mouse tails by the use of the following primers: C133 (5′-GCGTAAA TGGTTGATTGTCC-3′), W123 (5′-GATTG TCAGCACAGAAATCTTCCC-3′), and Mut2 (5′-GCGCTCCCCTACCCGGTAGATT-3′). PCR was performed with a mixture of all three primers. The C133/W123 combination generates a 256 bp wild-type band, and the C133/Mut2 combination generates a 150 bp mutant band. Location of PCR primers is indicated schematically in (a) Current Biology 2001 11, 105-109DOI: (10.1016/S0960-9822(01)00019-7)

Figure 2 Histological sections of embryos grown in utero to e7.5. E6.5 and e7.5 decidua from NBS1+/− intercrosses were sectioned and stained with hematoxylin and eosin. A midsagital section is shown for normal and presumed mutant embryos. Abbreviations are as follows: ee, embryonic ectoderm; xe, extraembryonic ectoderm; pac, preamniotic cavity. Scale bars represent 100 μm Current Biology 2001 11, 105-109DOI: (10.1016/S0960-9822(01)00019-7)

Figure 3 Defective growth of NBS1−/− blastocysts in vitro. (a) E3.5 blastocysts were isolated from NBS1+/− intercrosses and cultured in 96 well plates. Pictures were taken on a daily basis with a phase contrast microscope (Zeiss Axiovert 100) prior to genotyping by PCR. The upper panel shows growth of NBS1+/− blastocysts, and the lower panel is an example of NBS1−/− blastocyst outgrowth. Inner cell mass (ICM) and trophoblast giant cells (TGC) are indicated. The scale bar represents 100 μm. (b) Examples of PCR genotyping of blastocysts after 4 days in vitro culture. A nested PCR strategy was used. DNA preparation was by the incubation of individual blastocysts with 20 μl lysis buffer (100 mM KCl, 10 mM Tris HCl, 2.5 mM MgCl2, 0.45% Tween 20, 0.45% NP40, and 1 mg/ml proteinase K) for 4–5 hr at 55°C. DNA samples (3–5 μl) were used for genotyping. In the first round of PCR, a common primer, C1400 (5′-GGCTAAGA TGTATTGCTCCG-3′), a wild-type specific primer, W210 (5′- GGATCTGGGAAGCTAAAGTATG-3′), and a mutant primer, 3PNT (5′- TAAAGCGCATG CTCCAGACT-3′) were used. For the second round of PCR, primers Com133, W123, and Mut2 (indicated in Figure 1) were used to amplify both wild-type and mutant fragments Current Biology 2001 11, 105-109DOI: (10.1016/S0960-9822(01)00019-7)