Mice thrive without Cdk4 and Cdk2

Generation of Cdk4^−/−;Cdk2^lox/lox and Cdk4^−/−;Cdk2^−/− mice. (A) Targeting strategy. The targeting vector carries three frt sequences flanking exons 2 and 4 as well as a PGK-HygR cassette used for positive selection. The vector also contains a PGK-TK cassette used for negative selection. Recombinant ES cell clones containing a floxed Cdk2^lox allele were used to generate mice carrying the Cdk4^3frt and Cdk2^lox alleles in the same chromosome. These mice were sequentially crossed with transgenic mice expressing the Flpe (pCAG-Flpe) and Cre (CMV-Cre) recombinases to generate heterozygous Cdk4^−/−;Cdk2^−/− mice. Cdk4 coding sequences are indicated by hatched boxes. Cdk4 non-coding exons are indicated by open boxes. Cdk2 coding sequences are indicated by black boxes. Cdk2 non-coding exons are indicated by grey boxes. frt and loxP sites are indicated by open and closed triangles, respectively. Only the restriction sites used in these diagnostic hybridizations are indicated. The location of probes a and b used in Southern blot analysis is indicated by a thick line. (B) Southern blot analysis to identify the Cdk4^3frt recombinant allele. (C) Southern blot analysis to identify the Cdk4⁻ null allele.

Analysis of hematopoietic stem cells and lineage committed progenitors in late gestation embryos. (A) Cells were isolated from fetal livers of E17.5 embryos and analysed by flow cytometry. Relative percentages of hematopoietic stem cells (HSC), granulocyte-macrophage progenitors (GMP), common myeloid progenitors (CMP) and megakaryocyte-erythroid progenitors (MEP) are shown. (B) Left panel shows absolute cell numbers per fetal liver of wild type (empty boxes) and Cdk4^−/−;Cdk2^−/− (solid boxes) embryos. Right panel shows total numbers of GMP, CMP and MEP populations calculated per liver. Solid boxes represent samples obtained from Cdk4^−/−;Cdk2^−/− embryos and empty boxes represent wild type littermates. Error bars: SD.

Mice lacking Cdk4 and Cdk2 develop to term but die soon after birth due to cardiac failure. (Left) Representative images of Cdk4^+/+;Cdk2^+/+ (top) and Cdk4^−/−;Cdk2^−/− (bottom) P1 mice. Microscopic images from these mice showing (center left) H&E staining (×200) and (center right) Ki67 immunostaining of serial sections of ventricular walls (×200), and (right) H&E staining of the myocardium showing partially disorganized and enlarged cardiomyocytes with prominent capillaries (×400).

Biochemical characterization of cell cycle regulators in embryos lacking Cdk4 and Cdk2. (A) Expression levels of cell cycle regulators in wild type (Cdk4^+/+;Cdk2^+/+) and mutant (Cdk4^−/−;Cdk2^−/−) E17.5 embryos. Protein extracts were analyzed by immunoblotting with antibodies elicited against the indicated proteins. (B) Cyclin D2/Cdk6 and Cyclin E1–Cdk1 complexes were immunoprecipitated with antibodies against Cyclin D2 or Cyclin E1 and analyzed by immunoblotting using antisera against Cdk6 or Cdk1, respectively. M: mock immunoprecipitates; WCE: whole cell extract at 1:20 of starting material prior to immunoprecipitation. (C) Analysis of pRb phosphorylation levels using antibodies specific for phosphorylated residues S807/811, S780 or S608.

Proliferative properties of primary and immortal MEFs. (A) Left panel shows proliferation of primary MEFs at passage 2. Center panel shows immortalization of primary MEFs by continuous passage following a classical 3T3 protocol. Cdk4^+/+;Cdk2^+/+ (open circles) and Cdk4^−/−;Cdk2^−/− (filled circles). Left panel shows proliferation of immortal Cdk4^−/−;Cdk2^lox/lox MEFs infected with empty virus (open circles) or with virus expressing Cre recombinase (filled circles). Insert shows a Southern blot depicting complete cleavage of the Cdk2^lox allele in MEFs infected with virus expressing Cre recombinase (E, empty infected). (B) Percentage of quiescent Cdk4^+/+;Cdk2^+/+ (open bars) and Cdk4^−/−;Cdk2^−/− (filled bars) MEFs entering S phase upon addition of serum in the presence of 50μM BrdU. Top panel shows results with primary MEFs. Bottom panel shows results with immortal MEFs. Error bars: SD. (C) Expression of Cdk1, Cyclin A2 (Cyc A2), hypophosphorylated (Rb) and phosphorylated (P-Rb) forms of pRb in immortal MEFs at the indicated times after the addition of serum. β-Actin serves as loading control.

Generation of adult mice lacking Cdk4 and Cdk2. (A) Levels of excision of the conditional Cdk2^lox alleles in Cdk4^−/−;Cdk2^lox/lox;RERT^ert/ert exposed to 4OHT for 4 months. DNA was isolated from the indicated tissues of the resulting Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice and analyzed by Southern blot. The migration of the conditional Cdk2^lox and null Cdk2⁻ alleles is indicated by arrowheads. The percentage of recombined Cdk2⁻ allele in each of the tissues is indicated at the bottom. (B) Immunoblot analysis of the levels of expression Cdk4 and Cdk2 in some of the tissues shown in (A). As controls, we used extracts from the same tissues obtained from Cdk4^+/+;Cdk2^+/+;RERT^ert/ert mice treated with 4OHT. β-Actin served as loading control.

Normal adult homeostasis and cell proliferation levels in tissues of Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice. (A) Five-month-old Cdk4^+/+;Cdk2^+/+;RERT^ert/ert, Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert and Cdk4^−/−;Cdk2^lox/lox;RERT^ert/ert mice. The Cdk4^+/+;Cdk2^+/+;RERT^ert/ert and the Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice were treated with 4OHT, whereas the Cdk4^−/−;Cdk2^lox/lox;RERT^ert/ert mouse was treated with oil. (B) Ki67 immunostaining of the indicated tissues obtained from 5-month-old 4OHT-treated Cdk4^+/+;Cdk2^+/+;RERT^ert/ert and Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice. Esophagus: most of the stratum germinativum cells are proliferative (×400). Small intestine: proliferative cells are restricted to the lower part of the intestinal crypts (×200). Spleen: strong proliferation in the red pulp indicative of an active hematopoiesis. Most of the lymphoid cells in the white pulp are quiescent except those forming germinal centers (×50). Thymus: most thymocytes in the cortex are positive for Ki67 immunostaining (×100). The presence of the ert alleles in the description of the genotypes in the figure has been omitted for clarity.

Liver regeneration in Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice. (A) 4OHT-treated Cdk4^+/+;Cdk2^+/+;RERT^ert/ert and Cdk4^−/−;Cdk2^Δ/Δ;RERT^ert/ert mice were submitted to partial hepatectomy (PH) as indicated in Section 4 and the regenerated livers analyzed 9 days later by H&E and Ki67 immunostaining. A small hematopoietic cluster is indicated by arrowheads. (B) Southern blot analysis of the excision of conditional Cdk2^lox alleles in resected (Pre) and regenerated (Pos) liver samples from two independent Cdk4^−/−; Cdk2^Δ/Δ;RERT^ert/ert mice. DNAs from brain (Br) and testis (Ts) tissue from one of these mice were also tested as controls. These tissues are known to undergo limited cleavage of the Cdk2^lox allele (see Figure 5). (C) Immunoblot analysis of the expression of Cdk4, Cdk2 and Cdk1 in resected (Pre) and regenerated (Pos) livers of 4OHT-treated Cdk4^+/+;Cdk2^+/+;RERT^ert/ert and Cdk4^−/−; Cdk2^Δ/Δ;RERT^ert/ert mice. β-Actin served as loading control. The presence of the ert alleles in the description of the genotypes has been omitted for clarity.