| IntroductionCell cycle regulation and its association with cellular carcinogenesis are hot research areas in modern cell biology, in which the association, activation and disassociation of the complex composing cyclins and cyclin - dependent kinases (CDK) are the main molecular events underlying the regulatory process. CDK2, one of the important members in CDK family, directly regulates the critical steps in cell cycle progression including transition of G1/S checkpoint, DNA replication and centrosome duplication. DOC - 1R was identified in 1999 as a putative tumor suppressor gene. The encoded protein was showed to bind CDK2 in vitro and might be a specific inhibitor of CDK2. The DOC - 1R protein may prevent CDK2 from its nuclear import, which is served as one of the inevitable steps for CDK2 activation. In the present study, the effect of DOC - 1R expression on DNA replication and centrosome duplication was investigated using plas-mid DNA transfection and dual immunofluorescent microscopy. In addition, preliminary analysis of CDK2 sequence and its nuclear import was performed. Finally , pZX plasmids were constructed for labeling expression of transgene in situ which will be used in our future research work.Materials and Methods1. Construction of PEGFP-CDK2, PEGFP-CDK2N and pEGFP-CDK2CWild type and deletion mutants of CDK2 were amplified by polymerasechain reaction with pCMV - CDK2 plasmid DNA as template. After restrictiondigestion with HindlH and BamH I , the fragments were separated by 1% agar-ose gel, purified, and inserted into the linear pEGFP-C3 plasmid at the same re-striction sites using standard cloning techniques. The insert sequence, direction and open reading frajne in the resulted recombinant plasmids were confirmed by restriction and sequence analysis. The plasmid encoding wild type CDK2 was named pEGFP-CDK2, and the deletion mutants lacking the last and first 97 ami-no acids as pEGFP-CDK2N and pEGFP-CDK2C, respectively. The purified plasmid DNA samples were stored at - 20C for further transfection.2. Preliminary analysis of CDK2 sequence and its nuclear import Transient plasmid DNA transfection into HeLa cells was performed usingLIPOFECTAMINE鈩?PLUS Reagent according to the manufacture' s instruction. pEGFP-CDK2N and pEGFP-CDK2C were transfected the HeLa cells along with pEGFP-CDK2 as control. Each experiment was repeated at least three times. Serum - starved for 2 days post - transfection, fluorescent cells were viewed and photographed with Leica DMIRE2 fluorescent microscope equipped with FITC filter 4h after serum stimulation. DNA transfection into CHO cells was carried out as above. Two hours after transfection, HU was added- to a final concentration of 4mM for a 40 - hour - incubation. Fluorescent cells were then observed and photographed with Leica DMIRE2 fluorescent microscope.3. Construction of pZX plasmidsThe DNA segments of enhanced green fluorescent protein (EGFP), internal ribosome site (IRES) and a two - stranded ob'gonucleotide coding the C -terminal 20 amino acids of H - ras oncoprotein ( RASC20 ) were inserted into the multiple cloning sites of the eukaryotic expression vector pcDNAS. After sequence analysis of the new resulted plasmid pZX, liposome - mediated transfection was performed to HeLa cells with pEGFP - C3 as control. Then the subcel-lular localization of green fluorescent signals was detected after overnight incubation. Oligonucleotides containing EcoR I and EcoR V restriction sites were inserted into BamH I site of pZX to introduce more cloning sites.4. The effect of DOC - 1R expression on DNA replicationHeLa cells were transfected with pFLAG - DOC -1R plasmid DNA and incubated overnight. Transfected cells were subjected to indirect immunofluores-cent stains were BrdU incorporation for 1 hour. Fluorescent signals were then detected and photographed with Leica DMIRES fluorescent microscope equippedwith FITC and TRITC filters and x2 analysis was proceeded.5. The effect of DOC - 1R expression on centrosome dup...
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