Fig. 1.

Generation and analysis of Cdk1 conditional knockout mice and cells. (A) The Cdk1 genomic locus (I) was modified in ES cells with the targeting vector (II) shown. An FRT-flanked neomycin-selection cassette was introduced along with LoxP recombination sites (red triangles) on both sides of exon 3, which generated a mutant Cdk1 locus (III). For Southern blot analysis, 5′ and 3′ probes located outside of the targeting vector were used, after an EcoRV (RV) digest, resulting in a 31,206-bp (recombinant) and a 9,110-bp (5′) or 20,300-bp (3′) fragment for wild type (Fig. S1A). Upon expression of FLP recombinase, the neomycin cassette was removed, and only the LoxP sites remained in the locus (IV, Cdk1^FLOX). The levels of Cdk1 protein expression were similar in Cdk1^FLOX and Cdk1^WT mice (Fig. S1B). After Cre recombinase expression, exon 3 was excised (V), which resulted in deletion of Cdk1 and a frame shift. PCR genotyping primers are indicated (Pr1, -2, and -3), and the sequences can be found in Table S1. (B) To generate Cdk1 knockouts, Cdk1^FLOX mice were crossed with β-actin–Cre mice expressing Cre recombinase ubiquitously in all tissues, including germ line. The resulting Cdk1^WT/NULL mice were interbred, and the offspring was analyzed at weaning (P21), midgestation (E10.5), or blastocyst (E3.5) stage. (C and D) Cdk1^NULL/NULL blastocysts were visualized by Hoechst staining of their nuclei followed by fluorescence microscopy. Comparison of Cdk1-deficient blastocysts with heterozygous or wild-type littermates indicated a reduced number of cells (C); however, their nuclei were larger in size (D). (E and F) Three independent MEF lines (Fig. S1 C–H) were treated with 4-OHT to induce Cdk1 knockout, and their proliferative potential was monitored by 3T3 and alamarBlue proliferation assays for several passages or days, respectively (E). Deletion of Cdk1 resulted in a rapid arrest of cellular proliferation and premature onset of cellular senescence that was detected by senescence-associated β-gal staining (F). Therefore, cells lacking Cdk1 cannot proliferate but instead enter a senescent state and survive in culture medium.