Rescue of Embryonic Epithelium Reveals That the Homozygous Deletion of the Retinoblastoma Gene Confers Growth Factor Independence and Immortality but Does Not Influence Epithelial Differentiation or Tissue Morphogenesis
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m205361200
ISSN1083-351X
AutoresKathleen C. Day, Michael T. McCabe, Xin Zhao, Yuzhuo Wang, Joanne Davis, John L. Phillips, Marion von Geldern, Thomas Ried, Mark Kukuruga, Gerald R. Cunha, Simon W. Hayward, Mark L. Day,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb−/−) fetal mice has allowed for the isolation and characterization of the first Rb−/− prostate epithelial cell line. This cell line, designated Rb−/−PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb−/−PrE cultures. When Rb−/−PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb−/−PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb−/−PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb−/− prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb−/− fetuses. The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb−/−) fetal mice has allowed for the isolation and characterization of the first Rb−/− prostate epithelial cell line. This cell line, designated Rb−/−PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb−/−PrE cultures. When Rb−/−PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb−/−PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb−/−PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb−/− prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb−/− fetuses. The Rb 1The abbreviations used are: Rb, retinoblastoma; PCNA, proliferating cell nuclear antigen; rUGM, rat urogenital mesenchyme; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ER, estrogen receptor; DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride; SKY, spectral karyotype; CGH, comparative genomic hybridization; AR, androgen receptor; mDLP, murine, prostate-specific dorsal lateral protein marker; MEFs, mouse embryonic fibroblasts 1The abbreviations used are: Rb, retinoblastoma; PCNA, proliferating cell nuclear antigen; rUGM, rat urogenital mesenchyme; FBS, fetal bovine serum; PBS, phosphate-buffered saline; ER, estrogen receptor; DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride; SKY, spectral karyotype; CGH, comparative genomic hybridization; AR, androgen receptor; mDLP, murine, prostate-specific dorsal lateral protein marker; MEFs, mouse embryonic fibroblastsgene product mediates numerous cellular functions including cell cycle regulation (1Chen P.L. Scully P. 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The overlapping function of two structurally related family members, p107 and p130, represents an interactive mechanism in which pRb, p107, and p130 share the ability to regulate different members of the E2F family and thus a variety of target genes (13Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1955) Google Scholar, 15Mulligan G. Jacks T. Trends Genet. 1998; 14: 223-229Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 16Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4277) Google Scholar). Although structurally similar, there is growing evidence supporting distinct cellular functions for eachRb family member. All three proteins are differentially expressed during mouse development (17Jiang Z. Zacksenhaus E. Gallie B.L. Phillips R.A. Oncogene. 1997; 14: 1789-1797Crossref PubMed Scopus (118) Google Scholar), and their ability to initiate growth arrest is cell type-specific (18Zhu L. van den Heuvel S. Helin K. Fattaey A. 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Cell Growth Differ. 1996; 7: 1571-1578PubMed Google Scholar) that pRb is activated during androgen-stimulated epithelial proliferation and during androgen ablation-induced apoptosis. The pRb protein has also been shown to function as a transcriptional co-activator of the androgen receptor (25Yeh S. Miyamoto H. Nishimura K. Kang H. Ludlow J. Hsiao P. Wang C., Su, C. Chang C. Biochem. Biophys. Res. Commun. 1998; 248: 361-367Crossref PubMed Scopus (107) Google Scholar). Taken together, these results tentatively position Rb as a central mediator of androgen action controlling the differentiation, growth, and death of prostate epithelium; however, this hypothesis has not been tested. Cell culture models currently available to study epithelial physiology have a limited scope of relevance with regard to the pRb pathway. Many transformed prostate cell lines, such as DU145, already exhibit nonfunctional pRb due to mutation (26Blagosklonny M.V. Prabhu N.S. El-Deiry W.S. 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The use of viral oncogenes has provided substantial insight into the function of the Rb family members and their roles in regulating cell cycle, cell growth, and differentiation; however, viral oncogenes are promiscuous in their interactions with other cellular proteins and promote genomic instability making interpretation of these experimental models difficult. The chromosomal imbalances directly influenced by viral oncogenes have been identified as either random or nonrandom genetic events and include gross chromosomal translocations (31Tsao S.W. Wong N. Wang X. Liu Y. Wan T.S. Fung L.F. Lancaster W.D. Gregoire L. Wong Y.C. Cancer Genet. Cytogenet. 2001; 130: 141-149Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 32Cottage A. Dowen S. Roberts I. Pett M. Coleman N. Stanley M. Genes Chromosomes Cancer. 2001; 30: 72-79Crossref PubMed Scopus (34) Google Scholar, 33Wan T.S. Chan L.C. Ngan H.Y. Tsao S.W. Cancer Genet. Cytogenet. 1997; 95: 166-172Abstract Full Text PDF PubMed Scopus (23) Google Scholar). Therefore, models that specifically target and inactivate Rb, which minimize complicating genetic alterations inherent with viral oncogenes, might provide novel insight into the physiologic role of Rb in epithelial cells. The homozygous deletion of the Rb gene results in embryonic lethality due to a variety of developmental abnormalities (9Clarke A.R. Maandag E.R. van Roon M. van der Lugt N.M. van der Valk M. Hooper M.L. Berns A. te Riele H. Nature. 1992; 359: 328-330Crossref PubMed Scopus (886) Google Scholar, 10Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1505) Google Scholar, 11Lee E.Y. Chang C.Y., Hu, N. Wang Y.C. Lai C.C. Herrup K. Lee W.H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1113) Google Scholar). The embryonic lethality of the Rb knockout mutation has prevented the development of Rb−/− epithelial cell lines, and thus many of the functional aspects of pRb have not been independently characterized in this cellular population. Recently, the application of tissue recombination, using fetal tissue rescued fromRb−/− embryos and propagated in combination with wild type (Rb+/+) prostate stromal tissue, has enabled the development of Rb−/− prostate (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar, 35Phillips J.L. Hayward S.W. Wang Y. Vasselli J. Pavlovich C. Padilla-Nash H. Pezullo J.R. Ghadimi B.M. Grossfeld G.D. Rivera A. Linehan W.M. Cunha G.R. Ried T. Cancer Res. 2001; 61: 8143-8149PubMed Google Scholar, 36Hayward S.W. Wang Y. Cao M. Hom Y.K. Zhang B. Grossfeld G.D. Sudilovsky D. Cunha G.R. Cancer Res. 2001; 61: 8135-8142PubMed Google Scholar) and mammary grafts (37Robinson G.W. Wagner K.U. Hennighausen L. Oncogene. 2001; 20: 7115-7119Crossref PubMed Scopus (30) Google Scholar). In the current study, we describe the isolation and characterization of theRb−/− prostate epithelial cell line, Rb−/−PrE, derived from Rb−/− fetal urogenital precursor tissue rescued from embryonic mice (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar). To our knowledge, this is the first in vitro model to allow for the study of targeted Rbdeletion on an epithelial population and provides a unique experimental platform with which to investigate the physiological consequences ofRb deletion on the regulation of cell cycle, differentiation, cell survival, and carcinogenesis. Heterozygous Rb+/− mice were purchased from The Jackson Laboratories (Bar Harbor, ME). To increase litter size and thus the chances of Rb−/− offspring, the heterozygotes were crossed to CD1 mice. The genetic identity of the offspring was confirmed by PCR genotyping to identify the presence of the neomycin selection cassette that was used to disrupt the Rb gene. The Rb heterozygotes were crossed ant the fetuses rescued, and the prostatic ductal tips were then recombined as described previously (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar). The Rbstatus of tissue grafts was determined by PCR analysis. Control (The Jackson Laboratories) and experimental DNA samples were amplified using wild type- and mutant-specific primers and separated on 2% agarose gels containing ethidium bromide. The protocol for PCR cycling conditions was obtained from The Jackson Laboratory technical support ([email protected]). PCR primers (The Jackson Laboratories) used for genotyping tissues and cell lines were as follows:Rb knockout allele, forward 5′-AAT TGC GGC CGC ATC TGC ATC TTT ATC GC-3′ (oIMR025) and reverse 5′-GAA GAA CGA GAT CAG CAG-3′ (oIMR027); Rb wild type allele, forward 5′-AAT TGC GGC CGC ATC TGC ATC TTT ATC GC-3′ (oIMR025) and reverse 5′-CCC ATG TTC GGT CCC TAG-3′ (oIMR026) (10Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1505) Google Scholar). Serial tissue recombination was used to assess the ability of theRb−/− epithelial cells to undergo multiple rounds of growth and generate immortalized tissue. A ductal tip of ∼300 μm was micro-dissected from rescued Rb−/− prostatic tissue (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar), recombined with normal rat urogenital mesenchyme (rUGM), and grafted beneath the renal capsule of an intact male athymic mouse host. After 1 month of growth, the host was sacrificed, and the graft was retrieved. The resultant 40 mg of prostatic tissue (38Norman J.T. Cunha G.R. Sugimura Y. Prostate. 1986; 8: 209-220Crossref PubMed Scopus (54) Google Scholar) was again micro-dissected, and another 300-μm ductal tip was recombined with fresh rUGM and grafted into a new mouse host to produce a “second generation” graft. This recombination protocol, repeated 8 times, resulted in ∼13 epithelial population doublings for each round of recombination and re-grafting as estimated by the number of epithelial cells from the tissue weight using the Coffey equation 1g = 109 cells (39DeKlerk D.P. Coffey D.S. Investig. Urol. 1978; 16: 240-245PubMed Google Scholar). Rb−/− prostate grafts were established in nude mice, and then the ductal fragments were recombined with rUGM as described previously (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar). A portion of each excised graft was fixed for histological examination, and the rest were utilized to create Rb−/−PrE epithelial cultures. The tissue was minced with a scalpel and forceps and plated onto a tissue culture dish coated with collagen substrate in a minimal volume of medium to allow for attachment of the tissue to the matrix. These tissues were grown in RPMI 1640 (BioWhittaker). Culture media were supplemented with ITS (5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, Collaborative Research), BPE (10 μg/ml bovine pituitary extract, Sigma), epidermal growth factor (10 μg/ml, Collaborative Research), cholera toxin (1.0 μg/ml, Sigma), amphotericin B (250 μg/ml, fungizone, Invitrogen), dexamethasone (5 μm, Sigma), 200 mml-glutamine (Invitrogen), and 100 units/ml penicillin G and 100 units/ml streptomycin (BioWhittaker). This formulation supports the growth of epithelial cells while retarding the growth of the fibroblast cells. Approximately 10–14 days after plating, the tissue pieces were removed from the cultures, and selection was initiated with 200 μg/ml G418 (BioWhittaker). Once large areas of epithelial cells became established, cells were passaged 1:3 by trypsinization. Between passage 5 and passage 10, cultures were gradually switched to a medium containing only 5% FBS,l-glutamine (Invitrogen), and 100 units/ml penicillin G and 100 units/ml streptomycin (Invitrogen) in RPMI 1640 termed “5% FBS growth medium.” Wild type PrE cultures were generated from prostates excised from 6-week-old strain-matched male CD1 mice (Harlan Laboratories) following euthanasia with CO2. Prostate tissues were minced and plated in the defined BPE-containing culture medium described above. These cultures were maintained identical to the Rb−/−PrE described here without the addition of G418. The wild type control cell line, termed PrE, utilized for comparison herein, spontaneously immortalized in culture and was therefore utilized as a control for spontaneous immortalization of mouse prostatic epithelial cultures. Mouse Rb−/−PrE cells were grown on Falcon chamber slides to 70% confluence and fixed with 100% ethanol on ice for 5 min before washing with two changes of cold PBS. Fixed cells were then stained with Hoechst 33258 dye (5 μg/ml, Sigma) for 1 min at room temperature. Following staining, cultures were again washed three times in cold PBS, wet-mounted (Biomeda Corp.), and photographed using a Zeiss Axioskop fluorescent microscope to confirm that the cells were of mouse origin (40Cunha G.R. Vanderslice K.D. Stain Technol. 1984; 59: 7-12Crossref PubMed Scopus (73) Google Scholar). Wild type and Rb−/−PrE (5 × 105) cells were plated into 60-mm culture dishes containing 5% FBS growth medium. Three days after plating (termed “Day 0”), culture media were changed to serum-free media consisting of RPMI 1640 (BioWhittaker), 100 units/ml penicillin G, and 100 units/ml streptomycin (without phenol red and withoutl-glutamine). Cultures were fed with the aforementioned serum-free medium every 3 days and counted at the indicated times, where each time point is the average and S.D. of triplicate dishes. Viable cell counts were analyzed by trypan blue exclusion. Photographs were taken on a Nikon Diaphot 200 with a Nikon digital camera. At passage 21, Rb−/−PrE cells were utilized to generate prostate grafts via cellular recombination. To prepare grafts, 2.5 × 105 urogenital mesenchymal cells and 1 × 105 Rb−/−PrE cells were combined in a collagen matrix as described previously (41Hayward S.W. Haughney P.C. Lopes E.S. Danielpour D. Cunha G.R. Prostate. 1999; 39: 205-212Crossref PubMed Scopus (51) Google Scholar). These grafts were then transplanted beneath the renal capsule of an adult, male nude mouse host (Charles River Laboratories) and grown for 1 month. Host animals were then sacrificed, and the grafts were harvested, subjected to fixation, and evaluated utilizing immunohistochemical techniques. Tissue sections were deparaffinized in Histoclear (National Diagnostic) and hydrated in graded alcoholic solutions and PBS. Endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in methanol for 30 min and washed in PBS prior to staining. Immunocytochemical staining for expression of cytokeratins CK8, CK14, and CK18 in Rb−/−PrE cells was performed as described previously (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar). Cells were grown on chamber slides (Falcon) coated with 5 μg/ml fibronectin prior to fixation with 100% ethanol for 5 min on ice. Cells were then washed with PBS, and glucose oxidase substrate was employed in conjunction with the mouse and rabbit staining kits from Vector Laboratories. Staining for the AR and estrogen receptor α (ERα), mDLP, and p63 was also repeated with the re-grafted Rb−/−PrE cells (passage 20). Following growth in nude mouse hosts, the re-grafted tissue was harvested and subjected to the staining procedure as described previously (34Wang Y. Hayward S.W. Donjacour A.A. Young P. Jacks T. Sage J. Dahiya R. Cardiff R.D. Day M.L. Cunha G.R. Cancer Res. 2000; 60: 6008-6017PubMed Google Scholar,42Signoretti S. Waltregny D. Dilks J. Isaac B. Lin D. Garraway L. Yang A. Montironi R. McKeon F. Loda M. Am. J. Pathol. 2000; 157: 1769-1775Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). Primary antibodies were obtained as follows: anti-E-cadherin (Transduction Laboratories, C20820), anti-Rb (Pharmingen, 14001A), anti-p107 (Transduction Laboratories, R27020), anti-p130 (Santa Cruz Biotechnology, SC-318), anti-p21 (Pharmingen, 556430), anti-p53 (Oncogene, OP29), anti-AR (Santa Cruz Biotechnology, SC-816), anti-estrogen receptors α and β (Santa Cruz Biotechnology, SC-542 and SC-8974, respectively), anti-cyclin D1 (Santa Cruz Biotechnology, SC-8396), anti-cyclin E1 (Santa Cruz Biotechnology, SC-481), anti-PCNA (Santa Cruz Biotechnology, SC-9857), anti-actin (Santa Cruz Biotechnology, SC-1615), and high molecular weight pan-cytokeratin (Z0622, Dako). Horseradish peroxidase-conjugated secondary antibodies were obtained as follows: donkey anti-mouse (Amresco, E974), goat anti-rabbit (Bio-Rad), and donkey anti-goat (Bio-Rad). For protein analysis, cultured cells were lysed on ice in 50 mm Tris, pH 7.5, 120 mmNaCl, 0.5% Nonidet P-40, 1 mm EGTA, and protease inhibitors (40 μm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 50 μg/ml aprotinin, 200 μm sodium orthovanadate). Following centrifugation, the supernatants were collected, quantitated using a Bradford microtiter assay, and denatured with a reducing 2× sample loading buffer for 5 min at 100 °C. All proteins were then separated on Tris/glycine pre-cast NOVEX gels and analyzed utilizing the ECL detection system (Amersham Biosciences) as described previously (43Zhao X. Gschwend J.E. Powell C.T. Foster R.G. Day K.C. Day M.L. J. Biol. Chem. 1997; 272: 22751-22757Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Cultures of PrE and Rb−/− PrE were maintained in RPMI 1640 (BioWhittaker) containing 5% dextran-coated, charcoal stripped FBS, termed “5% CCS growth medium,” and compared with cultures grown in 5% FBS growth medium. For these experiments, PrE and Rb−/−PrE cells were plated at a density of 5 × 105 cells into 100-mm culture dishes, and viability and cell number were assessed via trypan blue (Invitrogen) exclusion on various days after plating. PrE and Rb−/−PrE (1 × 106, passages 15–20) were plated into 100-mm dishes and analyzed 4 days later to determine log phase cell cycle profiles. To ascertain growth arrest in confluent cultures, cells were plated at a higher density (2 × 106 cells per dish) and retained in culture for a total of 15 days. Serum-containing medium was replaced every 3 days on the long term cultures. Cells were harvested by trypsinization, fixed, and stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI). Prior to examination of cells by flow cytometry, 1 × 106 cells were collected by trypsinization, centrifuged, and reconstituted with 800 μl of PBS (without calcium or magnesium). One drop of an internal trout DNA control (Reiss Enterprise, 1007) was added to each sample. Then, 3.5 ml of cold 100% ethanol was added dropwise while mixing for fixation. Samples were then incubated for 1 h on ice prior to centrifugation and re-hydrated with 1 ml of PBS for 15 min on ice. Again, all samples were centrifuged and reconstituted with 1.5 ml of DAPI staining buffer (100 mm Tris, pH 7.4, 150 mm NaCl, 1 mm CaCl2, 0.5 mm MgCl2, and 0.1% Nonidet P-40). A final concentration of 1.0 μg/ml DAPI (Molecular Probes, D-1306) was utilized for flow cytometric evaluations. Flow cytometry was carried out at the University of Michigan Flow Cytometry Core facility using BD Biosciences FACSVantage SE model 127. Data were acquired to 105 events per sample. MultiCycle software (Phoenix Flow Systems, San Diego, CA) was utilized to estimate the percentage of cells in G1, S, and G2M phases of the cell cycle populations. Metaphase chromosomes from cultures of wild type PrE and Rb−/−PrE were obtained by mitotic shake off after 1 h of colcemide (1 μg/ml) treatment. Slides were hybridized with SKY kits, prepared from flow-sorted chromosomes, and detected 72 h later as described previously (44Difilippantonio M.J. Zhu J. Chen H.T. Meffre E. Nussenzweig M.C. Max E.E. Ried T. Nussenzweig A. Nature. 2000; 404: 510-514Crossref PubMed Scopus (468) Google Scholar). Images of 10–15 metaphase cells were acquired using a DMRXA microscope (Leica, Wetzlar, Germany) equipped with a custom-designed SKY-3 optical filter (Chroma Technology, Brattleboro, VT), a spectral cube, and a charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Analysis was performed with SkyView software (Applied Spectral Imaging, Ltd., Migdal Haemek, Israel) as described elsewhere (45Liyanage M. Coleman A. du Manoir S. Veldman T. McCormack S. Dickson R.B. Barlow C. Wynshaw-Boris A. Janz S. Wienberg J. Ferguson-Smith M.A. Schrock E. Ried T. Nat. Genet. 1996; 14: 312-315Crossref PubMed Scopus (247) Google Scholar). For CGH analysis, DNA was prepared under high salt conditions. Biotin-labeled DNA was derived from PrE and Rb−/−PrE cultures and co-precipitated with digoxigenin-labeled reference DNA obtained from sex- and strain-matched Rb+/−and/or Rb+/+ mice. DNA was hybridized to sex-matched normal murine (C57) lymphocyte metaphase chromosomes and detected, and images were acquired with Q-CGH software (Leica Imaging Systems, Cambridge, UK) (46Weaver Z.A. McCormack S.J. Liyanage M. du
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