M. Kasim Diril1, Chandrahas Koumar Ratnacaram1, V.C.Padmakumar3, Tiehua Du4, Martin Wasser4,6, Vincenzo Coppola3, Lino Tessarollo3, and Philipp Kaldis1,2
1 - Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, Proteos#3-09, Singapore 138673.
2 - Department of Biochemistry, National University of Singapore (NUS); Singapore 117597.
3 - National Cancer Institute, Mouse Cancer Genetics Program, NCI-Frederick, Bldg. 560, 1050 Boyles Street, Frederick, Maryland 21702-1201, USA.
4 - Bioinformatics Institute (BII), A*STAR (Agency for Science, Technology and Research), 30 Biopolis Street, Matrix, Singapore 138671.
5 - National Cancer Institute, Mouse Cancer Genetics Program, NCI-Frederick, Bldg. 560, 1050 Boyles Street, Frederick, Maryland 21702-1201, USA.
6 - Department of Biological Sciences, National University of Singapore (NUS); Singapore 117597.
Published in Proc Natl Acad Sci U S A. 21 Feb 2012. [Epub ahead of print]
Cyclin-dependent kinase 1 (Cdk1) is an archetypical kinase and a central regulator that drives cells through G2 phase and mitosis. Knockouts of Cdk2, Cdk3, Cdk4, or Cdk6 have resulted in viable mice, but the in vivo functions of Cdk1 have not been fully explored in mammals. Here we have generated a conditional-knockout mouse model to study the functions of Cdk1 in vivo. Ablation of Cdk1 leads to arrest of embryonic development around the blastocyst stage. Interestingly, liver-specific deletion of Cdk1 is well tolerated, and liver regeneration after partial hepatectomy is not impaired, indicating that regeneration can be driven by cell growth without cell division. The loss of Cdk1 does not affect S phase progression but results in DNA re-replication because of an increase in Cdk2/cyclin A2 activity. Unlike other Cdks, loss of Cdk1 in the liver confers complete resistance against tumorigenesis induced by activated Ras and silencing of p53.
(A) The Cdk1 genomic locus (I) was modified in ES cells using the targeting vector (II) shown. A 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). 5' and 3' probes located outside of the targeting vector were used for Southern blot analysis after an EcoRV (RV) digest, resulting in a 31,206bp (recombinant) and a 9,110bp (5') or 20,300bp (3') fragment for wild type. Upon expression of FLP recombinase, the neomycin cassette was removed and only the LoxP sites remained in the locus (IV, Cdk1FLOX). The levels of Cdk1 protein expression was similar in Cdk1FLOX and Cdk1WTmice. 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, 3) and the sequences can be found in the Supporting Information.
(B) To generate Cdk1 knockouts, Cdk1FLOX mice were crossed with β-actin-Cre mice expressing Cre recombinase ubiquitously in all tissues including germ line. The resulting Cdk1WT/NULL mice were interbred and the offspring was analyzed at weaning (P21), midgestation (E10.5), or blastocyst (E3.5) stage. Cdk1 knockout mice are non-viable and die very early during embryonic development.
(C, D) Cdk1NULL/NULL blastocysts were visualized by Hoechst staining of their nuclei followed byfluorescence 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, F) Three independent MEF lines were treated with 4-OHT to induce Cdk1 knockout and their proliferative potential was monitored by 3T3 and Alamar Blue 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-galactosidase staining (F). Therefore, cells lacking Cdk1 cannot proliferate but instead enter a senescent state and survive in culture medium.
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