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

Fu Y, Wang SQ, Liu YP, Wang GP, Wang JT, Gong SS

Department of Otorhinolaryngology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China; fyent@163.com.

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

Published 2 April 2008 in J Zhejiang Univ Sci B, 9(4): 299-305.
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