The Expression Of M-CSF In NIH3T3 Cell Nucleus And Its Effect On Cell Movement

Objectives:(1) To construct the eukaryotic expression vector pEGFP/M-CSF and observe localization of GFP-M-CSF fusion protein in NIH3T3 cells.(2) To construct M-CSF-expressing in nucleus vector (pCMV/M-CSF) and establish a cell line which stably express M-CSF in NIH3T3 cells nucleus.(3) To explore the effect of nuclear M-CSF on the cell movement and cytoskeleton in NIH3T3 cells.Methods:Function fragment of M-CSF cDNA was amplified by Polymerase chain reaction(PCR) and inserted to pEGFP-C1 to construct pEGFP/M-CSF by gene recombination. The recombinant gene was identified by PCR, restriction enzyme analysis and DNA sequencing. After transfecting pEGFP/M-CSF into NIH3T3 cells using liposome, the localization of M-CSF was detemined by detection of GFP with fluorescence microscopy. Then the active M-CSF fragment was subcloned into nucleus-localization expression vector pCMV/myc/nuc to construct pCMV/M-CSF, which will lead the protein into the cell nucleus. After verifying by PCR, restriction enzyme analysis and sequencing, the pCMV/M-CSF was transfected into NIH3T3 cells and the cells clones were selected with G418. The expression and localization of M-CSF in NIH3T3 cells were verified by RT-PCR, immunocytochemistry and Western blot. Finally, the effect of nuclear M-CSF on cell movement and cytoskeleton were analyzed by cell scratch assay and dying with Coomassie brilliant blue.Results:...