强力霉素调控小鼠E1A 激活基因阻遏子表达NIH3T3 细胞系的建立☆.docVIP

强力霉素调控小鼠E1A激活基因阻遏子表达NIH3T3细胞系的建立***☆ 韩雅玲，崔继福，康 建，张效林，闫承慧 Establishment of a mouse NIH3T3 cells line expressing cellular repressor of E1A-stimulated genes regulated by doxycycline Han Ya-ling, Cui Ji-fu, Kang Jian, Zhang Xiao-lin, Yan Cheng-hui Abstract BACKGROUND: Previous data show that cellular repressor of E1A-stimulated genes (CREG) regulates proliferation and phenotype switch of human vascular smooth muscle cells. Further research is needed to explore the relationships between effects on proliferation, differentiation and apoptosis of cells and dosage of CREG. OBJECTIVE: To establish a mouse NIH3T3 cells line whose expression of CREG is regulated by the addition of doxycycline, and to provide the basement for the study of biological function of CREG. DESIGN, TIME AND SETTING: A controlled observation was accomplished in Cardiovascular Institute of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA from May 2006 to March 2008. MATERIALS: RevTet-On system and 3T3 cell line were used in this study. METHODS: Mouse CREG (mCREG) cDNA duplicated by reverse transcription-polymerase chain reaction (RT-PCR) from mouse RNA was cloned into a retroviral response vector (pRevTRE) controlled by doxycycline responsive element. The recombinant vector (pRevTRE-mCREG) was constructed and identified by sequence analysis. NIH3T3 cells were transfected by RevTet-On and selected by G418. NIH3T3-RevTet-On clones were transfected by RevTRE-mCREG and selected by hygromycin. The expression of G418 and TRE was detected by RT-PCR analysis in double-stable transfection clones. MAIN OUTCOME MEASURES: The mCREG expression in cell lysate was evaluated by Western blotting after the NIH3T3-mCREG cells cultured in medium of different concentrations of doxycycline. RESULTS: The recombinant pRevTRE-mCREG was constructed successfully. The fragment inserted was confirmed by sequencing. The RevTet-On and RevTRE-mCREG was transferred into NIH3T3 cells and the clones