The Effect Of Nuclear M-CSF On The Cell Proliferation And The Identification Of M-CSF-interacting Proteins In Cos7 Cells

Objective1. To establish a cell line which stably express M-CSF in nucleus.2. To explore the effect of nuclear M-CSF on the proliferation of Cos7 cell, a African green monkey kidney cell line.3. To identify M-CSF-interacting proteins in Cos7 cells.Methods An active M-CSF fragment was amplified by Polymerase chain reaction(PCR) and subcloned to a eukaryotic expression vector (pCMV/myc/nuc) to construct a M-CSF-expressing in nucleus plasmid (pCMV/nuc/M-CSF) by gene recombination. The recombinant gene was identified by agarose gel electrophoresis, restriction enzyme analysis and sequencing. pCMV/nuc/myc, pCMV/nuc/GFP and pCMV/nuc/M-CSF were stably transfected into Cos7 cells using liposome. After screening with G418, the expression and localization of M-CSF in Cos7 cells were verified by fluorescence microscopy, RT-PCR, immunocytochemistry and Western blot. The effect of nuclear M-CSF on Cos7 cell proliferation was analyzed by cell count and MTT method. The M-CSF-interacting proteins were identified by poll-down test.Results The results showed that the size of inserted fragment in the recombinant gene corresponded to that of M-CSF, and that there was no reading frame shifts and mutations in recombinant M-CSF, and that green fluorescence protein only localized to the nucleus in Cos7 cells, which suggested that M-CSF expression vector in nucleus (pCMV/nuc/M-CSF) was successfully constructed. After transfecting pCMV/nuc/M-CSF into Cos7 cells by liposome and screening with G418, Our results indicated that the M-CSF-transfected Cos7 cells stably expressed both M-CSF mRNA and protein in nucleus, which suggested that a...