Transfection Of Neural Stem Cells With Green Fluorescent Protein Gene By Lipofectamine2000

Objective:To approach the optimization of Lipofectamine2000 mediated reporter enhanced green fluorescent protein(EGFP)gene transfection of neural stem cells(NSCs).To observe the effect of EGFP as a tracer for cell survival and differentiation after transfected NSCs were transplanted into striatum of normal rats,and provide experimental evidence for using NSCs as a resource of vehicle cells for gene therapy.Methods:(1)Neural stem cells were isolated from rat hippocampus,cultured and expanded by DMEM/F12 serum-free medium containing growth factors bFGF,EGF and B₂₇.The cells were passaged continuously by disassociating mechanically in order to purify and obtain the cells bulk.The cells' morphous was observed under inverted phase-contrast microscope,and flow cytometry was applied to estimate the proliferation of neural stem cells.Application growth curve estimate the capability of the cell.Immunocytochemistry was performed to detect the expression of Nestin,neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),and cyclic nucleotide phosphohydrolase(CNP).(2)At 37℃and 4℃,we culture NSCs in deifferent time,and then observe them.(3)Cotransfection of neural stem cells was conducted using fluorescent plasmid as a reporter gene with Lipofectamine2000.For establishing optimaltransfection conditions,we designed the techniques of determining the expression level of a reporter protein by varying parameters,such as differenttransfection time,different dose of lipofectamine,different dose of DNA,diffe-rent period of neural stem cells,and observed the expression of enhanc-ed green fluorescent protein(EGFP);Screening cell clones which stable expressing GFP,observing their property of growth.(4)Stable transfectants were stereotactically implanted into the strining of the living rats,observed the expression of enhanced green fluorescent pr-otein in vivo in 4,8,12,15 weeks.Results:(1)Immunocytochemistry study indicated that the neurospheres were Nestin-positive and could differentiate into multi-directions.Specific antigens of neurons,astrocytes,and oligodendrocytes were expressed also.(2)NSCs can be propagated under low temperature(4℃).(3)When transfection in the third-cultured hippocampus NSCs,the transfection time was 6 hours and the dose of lipofectamine(μl)was 8,the dose of DNA(μg)was 6,the eficency of was 27.6%with liposome transfection method,;3 behalf of the most efficient transfer,There is a certain relationship betewn transfer rate and the G2-M phase.G418 minimum lethal concentration is 500μg/ml,MTT assay of the same period of transfer and the untransfer NSCs, Activity between the two groups no significant difference(P＞0.05).(4)NSCs transfected with EGFP were able to express enhanced green fluorescent protein after implant into vivo in the 4 and 8 weeks,in the 15 weeks we weren't see andthing.Conclusions:(1)The cells isolated from rat hippocampus can only be characterized as stem cells of the central nervous system according to their undifferentiated features,the capacity of self-renewing,proliferation and pluripotentiality,and the expression of Nestin antigen.(2)We concluluded that the optimal transfection coditions of Lipofectamine2000 mediated reporter gene transfection.NSCs provide exprimental evidence for investigating gene transfer of NSCs;NSCs were the ideal vehicle cells of gene theray;NSCs transfected with EGFP were easily traced and observed for transplant study.