Volume 14, Issue 1, Pages 45-55 (January 2001) Analysis of C-MYC Function in Normal Cells via Conditional Gene-Targeted Mutation  Ignacio Moreno de Alboran, Rónán C O'Hagan, Frank Gärtner, Barbara Malynn, Laurie Davidson, Robert Rickert, Klaus Rajewsky, Ronald A DePinho, Frederick W Alt  Immunity  Volume 14, Issue 1, Pages 45-55 (January 2001) DOI: 10.1016/S1074-7613(01)00088-7

Figure 1 Deletion of c-myc from Mouse Embryonic Fibroblasts (A) c-myc genomic locus, targeting construct and in vitro Cre deletion of the selectable marker neomicin. In vitro deletion of the neo gene results in c-myc locus flanked by 2 loxp sites (fl, flox allele). (B) In vitro Cre-mediated deletion of c-myc in MEFs. Southern blot analysis of DNA from two independent lines of MEFs before (-) and after infection with GFP-Cre retrovirus. After infection, GFP-positive cells were sorted by FACS. As a control, a retroviral vector expressing GFP only was used to infect c-mycfl/fl fibroblasts. c-myc flox allele (fl, flox), c-myc deleted allele (Δ). DNA was digested with EcoRI and hybridized with probe I (A). (C) Growth curve of wt c-mycfl/fl, and c-mycΔ/Δ MEFs. Wt MEFs were infected either with Cre-GFP (wt + Cre) or control GFP retrovirus (wt + GFP). c–myc-deficient (c-mycΔ/Δ) MEFs, obtained as described in (B), or c-mycfl/fl MEFs were infected with a Myc expressing retrovirus (c-mycfl/fl + Myc, and c-mycΔ/Δ + Myc). Four independent experiments are shown. Each experiment represents independent MEF lines. MEFs were harvested and plated at 2 × 105/10 cm dish. Duplicate plates were counted at the time points shown. (D) Cell cycle analysis of c-Myc-deficient MEFs (c-mycΔ/Δ). Cells were cultured for 48 hr and stained with Propidium iodide for DNA content and analyzed by FACS. Profile shown is representative of two independent experiments. (E) Proliferation rates of MEF lines generated as in (A). Proliferation was determined using a WST-1 cleavage assay (Boehringer Mannheim). Results shown are the average of two independent experiments performed in triplicate. (F) Western blot analysis on c-myc deleted (c-mycΔ/Δ) and nondeleted c-mycfl/fl MEFs, and of wt MEFs infected with Cre or GFP expressing retrovirus. The same amount of protein extract was used from all MEF lines. Immunoblot shown is representative of two independent experiments. Rb, retinoblastoma protein; hypo, hypophosphorylated; and hyper, hyperphosphorylated. Immunity 2001 14, 45-55DOI: (10.1016/S1074-7613(01)00088-7)

Figure 2 Deletion of c-myc from B Cells In Vivo (A) Specific c-myc deletion in B lymphocytes in vivo. Southern blot of DNA from different organs of c-mycfl/fl; CD19cre and thymus of c-myc+/fl; CD19cre mice. (B) Number of B and T cells in the spleens of wt, heterozygous c-myc+/fl; CD19cre mice, and homozygous c-mycfl/fl; CD19cre 3- to 4-week-old mice. The average of three mice of each genotype is shown. (C ) Southern blot of sorted mature B cells from spleen of a c-myc+/+; CD19cre or a heterozygous c-myc+/fl ; CD19cre mouse and a pool of 3–4 homozygous c-mycfl/fl; CD19cre mice at 0 24, 48, 72, and 96 hr after activation with anti-CD40 antibody and interleukin 4. Each panel is representative of two experiments with identical results. DNAs were processed as Figure 1B. c-myc flox allele (fl). (D) Thymidine incorporation upon anti-CD40 plus IL-4 stimulation of purified B cells from wt mice (no Cre expression) or heterozygous c-myc+/fl; CD19cre and homozygous c-mycfl/fl; CD19cre mice that express Cre (upper panels and lower left panel) and homozygous c-mycfl/fl mice that do not express Cre (lower right panel). (E) c-myc Southern blot of purified wt and heterozygous c-mycfl/+ ; CD19cre B cells indicating that Cre expression does not inhibit proliferation of c-mycfl/+ B cells. Purified mature wt and heterozygous B cells were mixed at 1:1 ratio and activated with anti-CD40 and IL-4 for the indicated times. No enrichment was observed for either wt or deleted (Δ) band after 3 days of culture. Experiment shown is representative of two independent experiments. Immunity 2001 14, 45-55DOI: (10.1016/S1074-7613(01)00088-7)

Figure 3 Failure of c-Myc-Deficient B Cells to Undergo Activation (A) Forward versus side scatter analysis of sorted mature B cells from spleen of a wt, a heterozygous c-myc+/fl; CD19cre and a pool of 3–4 homozygous c-mycfl/fl; CD19cre mice (3–4 weeks old). Cells were activated with anti-CD40 antibody and interleukin 4, and FACS analysis was performed at 0 and 48 hr. (B) c-myc-deficient B cells (Δ/Δ) are localized in gate (R). Activated B cells were purified from c-mycfl/fl; CD19cre mice, and PCR analysis was performed to detect the presence of the deleted (Δ-PCR) and nondeleted (fl-PCR) c-myc gene. After 48 hr after activation with anti-CD40 antibody, and interleukin 4 cells were sorted by FACS using gates R or A as indicated in (A) before performing PCR. Mouse embryonic fibroblasts, DNA from undeleted (MEF fl), or deleted (MEF Δ) and amplification of the constant region of IgM served as controls. Immunity 2001 14, 45-55DOI: (10.1016/S1074-7613(01)00088-7)

Figure 4 Anti-CD40-Treated c-Myc-Deficient B Cells Express Early Activation Markers but Not CD95 or CD95L (A) FACS analysis of surface expression of CD69, CD23, and B7.2 from anti-CD40 and interleukin 4 activated B cells (48 hr) from wt, a heterozygous c-myc+/fl; CD19cre and a pool of 3–4 homozygous c-mycfl/fl; CD19cre mice (3–4 weeks old). Δ/Δ and flox/flox cells were distinguished using gates R and A, respectively, as in Figure 3A. (B) FACS analysis of surface expression of CD95 and CD95L. Cells were activated as (A). Unstimulated cells were used as a negative control (shaded area) in all stainings. Results are representative of three independent experiments. c-myc flox allele (fl). Immunity 2001 14, 45-55DOI: (10.1016/S1074-7613(01)00088-7)

Figure 5 c-Myc-Deficient Mature B Cells Are Blocked at G0/G1 Stage of the Cell Cycle following Anti-CD40 Stimulation (A) Cell cycle analysis by FACS from sorted mature B cells from spleen of a wt, a heterozygous c-myc+/fl; CD19cre, and a pool of 3–4 homozygous c-mycfl/fl; CD19cre mice. Cells were activated for 48 hr as in Figure 3A and stained with propidium iodide (PI). (B) c-Myc-deficient cells are resistant to etoposide treatment. Southern blot of sorted mature B cells from c-mycfl/fl; CD19cre mice activated for 24 hr as in Figure 3A before being treated with etoposide (10 μM) for an additional 24 hr. In a second experiment, the ratio Δ/flox changed from ∼30/70 to 70/30 after etoposide treatment. Quantification was performed with Phosphorimager. (C) Western blot analysis on B220+ sorted B cells from c-mycfl/fl; CD19cre and wt mice activated as (A) for 10 hr. Rb, retinoblastoma protein. Actin is used as a loading control for Rb, p27, and c-Myc immunoblots. α-tubulin is used as a loading control for p21. Hypo, hypophosphorylated; and hyper, hyperphosphorylated. Experiment shown is representative of two independent experiments. (D) CDK2 and Cyclin D1-associated kinase activities are inhibited in c-My c-deficient B cells. The same amounts of protein extract from activated B cells (10–12 hr) from wt and c-mycfl/fl; CD19-Cre mice were immunoprecipitated with antibodies to either Cdk2 or Cyclin D1 and assayed for their ability to phosphorylate a GST-Rb substrate. The c-Myc immunoblot for the Cdk2 panel controls for the extent of deletion of c-myc within the B cell population used for the Cdk2 kinase assay. As the same extracts were used for the experiments in (C) and the CyclinD1 assay, the c-Myc immunoblot in (C) serves as the cMyc deletion control for the Cyclin D1 panel. Experiments illustrated are representative of two or three independent experiments employing different samples. c-myc flox allele (fl, flox). Immunity 2001 14, 45-55DOI: (10.1016/S1074-7613(01)00088-7)