Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Targeted disruption of murine Fancd2–/– gene. (A) Schematic of targeting vector, wild-type Fancd2 locus, targeted allele with neo–ura cassette, and targeted allele without neo–ura cassette. Homologous integration of the targeting plasmid replaces Fancd2 exons 26 and 27with a neo–ura cassette flanked by lox sites. The binding site for the probe is indicated by a black bar. (B) Southern blot analysis using genomic DNA from targeted ES cell clones digested with XbaI. Homologous integration of the targeting plasmid eliminates an XbaI site in intron 26, resulting in a nontargeted band at 3.6 kb and a wild-type band at 8.3 kb. (C) PCR genotyping using genomic DNA from wild-type, heterozygous, and mutant tail as templates. Mutant Fancd2 alleles with and without the neo–ura cassette are shown. The mutant band is 556 bp (with neo–ura) or 459 bp (without neo–ura). The wild-type band is 303 bp. (D) Western blot with anti-Fancd2 antibody on protein lysate from Fancd2 mutant, Fanca/c double mutant, wild-type, and Fanca mutant testes. Fancd2-S (155 kD) and Fancd2-L (162 kD) are visible in wild-type extracts. Only Fancd2-S is detectable in Fanca/c mutant and Fanca mutant extracts. No signal is detectable in Fand2 mutant extract. (E) Western blot with anti-Fancd2 antibody on protein lysate from wild-type and Fancd2 mutant MEFs either untreated or irradiated with 10 Gy. Fancd2 (155/162 kD) is detected in wild-type extracts but absent from Fancd2 mutant extracts. The absence of a band at 88 kD, corresponding to the predicted size of truncated Fancd2, is indicated.

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Disruption, Plasmid Preparation, Binding Assay, Southern Blot, Clone Assay, Mutagenesis, Western Blot, Irradiation

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Germ-cell and meiotic pairing abnormalities in Fancd2–/– mice. (A,B) H&E staining of testis sections from 7-week-old wild-type (A) or Fancd2–/– (B) littermates. All stages of spermatogenesis are seen in the wild-type adult testes. Fancd2–/– testes show a mosaic pattern of normal-appearing tubules (tubule 1), tubules with vacuolated Sertoli cell cytoplasm and no germ cells (tubule 2) and tubules with vacuolated Sertoli cell cytoplasm and decreased numbers of spermatocytes and spermatids (tubule 3). Magnification, 20×. (C,D) TUNEL staining of testes sections from 7-week-old wild-type (C) and Fancd2–/– (D) littermates. Occasional cells were labeled in wild-type tubules near the basement membrane, whereas Fancd2–/– testes contain tubules with multiple numbers of apoptotic spermatocytes. The nuclei of apoptotic cells are TUNEL-labeled with Cy3-dCTP and fluoresce red. Magnification, 20×. (E–H) Meiotic pairing abnormalities in Fancd2–/– spermatocytes detected by indirect immunofluorescence staining for Scp3 (green) and Rad51 (red). (E) Zygotene wild-type nucleus. (F) Zygotene Fancd2–/– nucleus with unusually long unpaired axial elements. (G) Late zygotene Fancd2–/– nucleus with multiple synaptic abnormalities. (H) Pachytene Fancd2–/– nucleus with unpaired (open arrowheads) and mispaired homologs (closed arrowheads). Magnification, 100×.

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Staining, TUNEL Assay, Labeling, Membrane, Immunofluorescence

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Fancd2 mutants are predisposed to microphthalmia. (A) Fancd2 mutants are smaller than wild-type controls at postnatal day 1. Microphthalmia is visible in Fancd2 mutants. (B) Histology of eye from a wild-type control pup (postnatal day 1). Magnification, 50×. (C) Representative histology of eye from a Fancd2–/– pup (postnatal day 1) shows a disorganized retina with lack of a distinct chamber or lens development. Magnification, 100×.

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Control

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Histology of carcinomas and survival curves for Fancd2 mutants and controls. Tumor-free (A; both adenoma and carcinoma) and carcinoma-free (B; only carcinoma) survival curves are shown for the combination of mixed and 129S4 populations. P values were determined by Logrank test. (C–F) H&E-stained paraffin-embedded sections of tumors from Fancd2–/– animals. Arrows indicate mitotic cells with condensed chromatin. Magnification, 200×. (C) Ovarian adenocarcinoma with large acini lined by tall columnar cells having abundant eosinophilic cytoplasm and highly pleomorphic hyperchromatic nuclei (129S4, 3 mo). (D) Mammary adenocarcinoma in which large clusters of mitotically active and pleomorphic cells were found, ranging from columnar cells to nests of small, undifferentiated cells in clusters (mixed, 13 mo). (E) Lung adenocarcinoma of broncho-alveolar origin, with rows of columnar epithelial cells having a high nucleus-to-cytoplasm ratio lining septa, sometimes forming papillae or filling air spaces (129S4, 13 mo). (F) Hepatocellular carcinoma is multinodular and well differentiated. The neoplastic cells vary from being smaller than normal hepatocytes to 2×–3× larger (mixed background, 18 mo).

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Staining

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Increased incidence of tumors of epithelial cell origin in Fancd2 mutant mice

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Mutagenesis

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: DNA damage sensitivity and S-phase checkpoint in Fancd2–/– MEFs and mice. Primary MEFs were challenged with increasing doses of mitomycin C (A), 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) plus UVA (PUVA; B), or ionizing radiation (C). (•) Wild-type control MEFs; (○) Fancd2 mutant MEFs; (▴) Fancc mutant MEFs. Experiments were performed in quadruplicate. Each data point represents the mean ± standard error of the mean (S.E.M.). (D) Survival curve of control (▪, •) and Fancd2 mutant (□, ○) mice following a dose of 9 (solid line) and 10 (broken line) Gy of whole-body irradiation. (E,F) Primary Fancd2–/– MEFs have an intact S-phase checkpoint. DNA synthesis was measured 30 min after treatment with 10 Gy of IR (E) or 0.5 ng/mL H-methoxy-tripsoralen (F) and is represented as the percentage of untreated controls. As a control, cells were also pretreated with 10 mM caffeine (caff). Experiments were performed in quintuplet. Error bars indicate the S.E.M.

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: Control, Mutagenesis, Irradiation, DNA Synthesis

Journal:

Article Title: Epithelial cancer in Fanconi anemia complementation group D2 ( Fancd2 ) knockout mice

doi: 10.1101/gad.1103403

Figure Lengend Snippet: Interaction of Rad51/Brca2 and Rad51 foci formation. (A) Whole-cell lysates from mouse testis of the indicated genotypes were separated by SDS-PAGE and probed with an antibody against Rad51. (B) Anti-Brca2 antibodies were used to immunoprecipitate protein complexes. Complexes were separated by SDS-PAGE and probed with an antibody against Rad51. “sn” indicates supernatant from immunocomplexes to compare bound versus unbound fraction. (C) Rad51 foci formation in wild-type and Fancd2–/– MEFs either untreated (–IR) or following 12 Gy of ionizing radiation (+IR). Cell nuclei were counterstained with DAPI (blue).

Article Snippet: The proteins were transferred to Immobilon-P Transfer membranes (Millipore Corp.) and Fancd2 was detected using a 1:5000 (testes) or 1:1000 (MEFs) dilution of anti-FANCD2 polyclonal antibody and 1:10,000 dilution of anti-rabbit secondary antibody (Amersham Life Science) and the ECL system (Amersham Life Science).

Techniques: SDS Page