Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Abstract

To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. We have successfully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.