Creation of TKO ES cells and MEFs. (A) Targeting of the first Rb allele in p107^−/−;p130^−/− ES cells inserted a stop codon (*) in Rb exon 3 and a hygro cassette in intron 3, creating a null allele (Rb^×3t). For the second allele, a targeting vector carrying a puro cassette and loxP sites (open triangles) was used (Rb^×3loxPuro allele). Upon introduction of Cre, Rb exon 3 was deleted, creating a null allele (Rb^×3Δ). The boxes marked E and I represent the two probes used in the Southern blot analysis, and the arrows marked 1, 2, and 3 represent the primers used in the PCR analysis. N, Nco1; P, Pst1; K, Kpn1; S, Sal1. Not to scale. (B) Southern blot analysis of TKO ES cells. Genomic DNAs of p107^−/−;p130^−/− ES cells with various Rb alleles were digested with Nco1 and Sal1 and hybridized with probe E (left panel) or with Pst1 and Kpn1 and hybridized with probe I (right panel). Arrows indicate alleles. The ES cells are on a mixed C57BL/6x129Sv genetic background, and the Nco1–Sal1 digest provides a polymorphism allowing us to distinguish both Rb alleles. (C) TKO MEF populations purity assayed by PCR. Primers 1–2 and 1–3 amplify Rb wild-type (wt) and mutant (mut) alleles, respectively (). At passage 0 (P0), TKO MEFs are mixed with wild-type MEFs. After selection, at passage 2 (P2), contaminating wild-type cells are absent. Three representative examples of TKO MEF populations are shown, as well as amplification results from control ES cells DNAs. (D) Western blot analysis of the three pRB family members in MEFs bearing different combinations of mutant alleles in Rb, p107, and p130. Two TKO MEF lines from two independent ES cells clones are shown. (E) E2F electromobility shift assays using extracts from exponentially growing TKO and control MEFs. p107 and p130 antibodies shift p107:E2F and p130:E2F complexes in Rb^−/− cells, and pRB antibody shifts pRB:E2F complexes in p107^−/−;p130^−/− cells. In contrast, in TKO cells, no complex is visible or shifted by these antibodies, and only the E2F-4 antibody shifts free E2F.* indicates a nonspecific complex. The probe has been run off the gel for better resolution. Bound and free (in complexes with pRB family or not) E2F are indicated as well as supershifts. When indicated, a 1000 times excess of specific competitor was added in the reactions.

Cell cycle analysis of TKO and control MEFs. (A) Flow cytometric cell cycle analysis of representative wild-type (wt), Rb^−/−, p107^−/−;p130^−/−, and TKO MEF cultures. G₁, S, and G₂/M populations are indicated as percentages of the whole population. Numbers correspond to the average of at least five independent experiments, with standard deviations (+/−). (B) Proliferation curves of TKO and control MEFs. The curves represent the average of three experiments for control MEFs and five for TKO MEFs, with standard deviations (error bars). (C) Cell size analysis of TKO and control MEFs. Asynchronously proliferating cells fixed and stained with PI were analyzed by flow cytometry. Populations were gated in a FSC-H by FL2-A dot plot and scored in a FSC-H histogram. The top panel represents whole populations and the two bottom panels 2n and 4n populations, as indicated.

Response of TKO and control MEFs to DNA damaging agents. (A) Cell cycle analysis of TKO and control MEFs following γ irradiation. Cell populations were sorted by flow cytometry in G₁, S, and G₂/M phases 18 h after irradiation. Numbers are relative to numbers obtained for nonirradiated cells, and standard deviations are indicated (error bars). (B) Cell cycle analysis of TKO and control MEFs following doxorubicin (dox) treatment. Representative flow cytometry results are shown for all the genotypes 18 h after treatment. The experiment was repeated at least three times with similar results. Panels for treated (+dox) and untreated (−dox) cells are aligned to show the variations in the G₁, S, and G₂/M populations. (C) Western blot analysis of p53 levels in exponentially growing TKO and control cells. (D) Northern blot analysis of p21^CIP1 expression in MEFs from different genotypes following DNA damage. RNAs were prepared from cells treated/not treated with doxorubicin (+/− dox). After hybridization with a p21 cDNA probe, the same blot was rehybridized with a Gapdh probe to control loading.

Response of TKO and control MEFs to cell–cell contact and low serum conditions. (A) Cell cycle analysis of confluent TKO and control MEFs. One or two representative results are shown for each genotype. The percentage of cells in the S phase of the cell cycle is indicated, with standard deviations (+/−). At least three experiments were performed. (B) Cell cycle analysis of confluent TKO and control MEFs in 0.1% serum. The percentages of cells in the S phase of the cell cycle are indicated, with standard deviations (+/−). At least three experiments were performed. “TKO” represents an experiment where all the cells, attached to the plate or floating, were included in the assay. No S-phase count was calculated in these conditions given the high sub-G₁ population. Control cells were analyzed in the same way. “TKO (attached)” corresponds to an experiment where floating cells were removed before cell cycle analysis. (C) Western blot analysis of the cleaved form of PARP specific of apoptotic cells in confluent TKO and control cells grown in 0.1% serum. Extracts were prepared from whole populations, including dead, floating cells. (D) Survival of confluent MEF populations grown in 0.1% serum. Annexin negative and positive cell populations were sorted by flow cytometry, and annexin negative cells were considered to be alive. Percentages are the average of at least three experiments, with standard deviations (error bars).

Immortality and partially transformed phenotype of TKO MEF populations. (A) Cultures derived from individual embryos were cultivated according to the 3T3 protocol. The graph shows the accumulated number of doublings that representative cultures have undergone. Full legend is as indicated in the graph. Groups of MEFs with the same genotypes or phenotypes are also indicated (CTRL, controls: wt, Rb^−/− and p107^−/−;p130^−/−). (B) Western blot analysis of p53 and of p16^INK4A, p19^ARF, and p21^CIP1 cell cycle inhibitors in early and late passage TKO clones. TKO cells (1–5) extracts were prepared at different passages, as indicated. Equal quantities of proteins were analyzed. (C) Proliferation of TKO and control cells after plating at low density. 1.3 × 10³ cells of each genotype were plated per 10-cm plate, and after 2 wk, colonies were stained and counted. At least five experiments were performed; average numbers and standard deviations (error bars) are shown. (D) TKO cells were infected with an empty retrovirus (pMIG) or with the same retrovirus expressing a human RB cDNA (pMIG-RB) and then plated at low density. The plates were stained with Giemsa to visualize the colonies 2 wk after plating. (E) Representative examples of TKO and control MEFs grown for 2 wk at confluency. TKO cells form a very dense layer with foci growing on top of it and with a tendency to peel off the plate. This phenomenon was not observed with wt, Rb^−/−, p107^−/−;p130^−/−, and p53^−/− cells. (F) Ability of MEFs with different genotypes to form colonies in soft agar. Pictures were taken at the same magnification (bar, 400 μm) after 24 d in culture.

Properties of H-RasV12-TKO MEFs. (A) Morphological changes induced by activated Ras in TKO MEFs. Left panel shows TKO MEFs infected with a pBabe empty retroviral vector, and right panel shows the same TKO MEFs clone infected with a pBabe-H-RasV12 vector. Photomicrographs were taken 2 wk after infection and selection (magnification 100×). No indications of premature senescence are observed in H-RasV12-TKO cells. (B) Representative proliferation curves of H-RasV12-TKO MEFs. H-RasV12-TKO MEFs continue to proliferate 2 wk after infection and selection, whereas under similar conditions H-RasV12 control MEFs (wild-type, Rb^−/− and p107^−/−;p130^−/−) undergo senescence (data not shown). (C,D) Representative examples of the enhanced ability of H-RasV12-TKO cells to form colonies 2 wk after plating at low density (C) or in soft agar (D), compared to TKO cells infected with a pBabe empty vector (TKO; bar 400 μm).