Doc-1r Gene On Cell Proliferation Cycle

IntroductionCell cycles regulation and relation with tumor are a hotpoint research in the field of cell biology now. Formation, activition and degradation of cyclin - dependent kinase(CDK) and cyclin complex are the main molecular foundation. CDK2 is an important member in CDKs family, it directly participates regulation of G^S checkpoint and DNA replication in S phase, its activity and functional regulation is one of the crucial key in cell cycle's operation, otherwise; CDK2 has very close relation with cell canceration. DOC - 1R gene is an candidate tumor suppressor gene which was discovered by zhangxue. It can interact with CDK2 specifically and restrain CDK2 transportation into the nucleus, which will block the formation of CDK2 and cyclin complex and hinder the process of cell cycle. Therefore, DOC - 1R gene may be an CDK2 inhibitor specifically. We have chosen DOC - 1R gene to construct the antisense plasmid and want to know its function on cell cycle. Otherwise; we have used immunoprecipitation and im-munofluorescence microscope technology to study the interaction with DOC - 1R and CDK2 proteins under physiological condition in cells, we attempt to find the evidence of interaction with DOC - 1R and CDK2 proteins, which will offer experiment basis for understanding the accurate role for DOC -1R gene in cell cycle S regulation.Materials and methods1. Mouse DOC - 1R genes screening and clone plasmid s obtaining Reference of human DOC - IRs cDNA sequence, we search the EST database in GenBank and choose an EST sequence of mouse DOC - 1R gene with having the greatest possibility to get the encoding cDNA sequence, named IMAGE 1921378. Designing primer to compose the sequence of mouse DOC - 1R cDNA. Application of biological informatics means, we have analyzed the protein sequence deduced from the encoding DOC - 1R gene and compared homolo-gy with other molecules. Then we have cloned the mouse DOC - 1R cDNA sequence into the pMEISS - FL3 plasmid.2. Reconstruction of antisense pcDNAS - DOC - 1R plasmidAfter transformation the pMEISS - FL3 - DOC - 1R plasmid into E. coli cells, we have extracted the plasmid and cut DOC - 1R fragment using Xho I to get a 975 bp DOC - 1R cDNA fragment. After recovering fragment and cutting it by Not I, we assay the sequence by electrophoresis. According to the mutliclone site of pcDNAS plasmid, we have used Xho I to cut pcDNAS plasmid and make its linearized. The pcDNAS and DOC -1R fragments have been reconstructed to form a new plasmid used by T4DNA ligation enzyme on 25C. After transformation, we have chosen the positive bacterium clones by bacterium colon PCR. With DNA sequencing, we have made sure direction of DNA sequence which inserted into the plasmid and have named sense and antisense reconstruction plas-mids respectively.3. Transfection of antisense pcDNA3 - DOC - 1R plasmid and testing influence on cell growthwe have transfected sense and antisense pcDNA3 - DOC - 1R plasmids into mouse NIH3T3 cells respectively, then transplanted cells which were in logarithm growth phase into 24 orifice plates, sampled one time per 24hs, every time chosen three orifices' cells. After counting, we have drawn cell growth curve. Preparation incubation media contained 1.7% agar and taking suitable quantity to lay on the bottom of the culture dish, we also have taken suitable quantity again contained 0.9% agar to lay on the top of dish and cultured transfected cells for 3 - 4 weeks, then observed numbers and sizes of colon formed by sense and antisense pcDNA3 - DOC - 1R plasmid respectively. After transfected by sense and antisense pcDNA3 - DOC -1R plasmid, we have also cultured mouse NIH3T3 cells with DMEM culture media containing G418 (400ug/ml) for two weeks. Using microscope, we have observed the plasmids'effection on a-bility of formation.4. Interaction with DOC - 1R and CDK2 proteins under physiological conditionUsing cotransfection,coimmunoprecipitation and western blotting, we have transfected the plasmids into 293 cells according to following...