AIMTo investigate the abilities of recombinant adeno-associated virus type 2 (rAAV2) transfecting neurospheres.

METHODSThe rAAV2 conjugated with FITC (rAAV2-FITC) was added into the culture medium of neurospheres and 30 minutes later the neurospheres were detected with a fluorescence microscopy to determine if the AAV can combine with neurospheres. The rAAV2 containing GFP reporter gene (rAAV2-GFP) was incubated with the neurospheres for a month and then detected the ability of transfecting neurospheres. The neurospheres transfected with rAAV2-containing GFP were transplanted to the brain of rats. A month later the rats were sacrificed and the brains were removed to detect if there are expressions of the reporter gene. The neurospheres were transfected with rAAV2 containing hypoxia responds elements (HRE) and vascular endothelium growth factor(VEGF) gene and reporter gene GFP (rAAV2-HRE-VEGF-GFP) and then cultured in low oxygen density environments. Seventy-two hours later the neurospheres were detected through a fluorescence microscopy.

RESULTSThe neurospheres incubated with rAAV2-FITC present bright green fluorescence. GFP, the reporter gene, can be seen clearly 1 month after being transfected with rAAV2-GFP. The same green fluorescence protein can be observed ex vivo as well. The fluorescence can be seen in neurospheres transfected by rAAV2-HREVEGF-GFP only in low oxygen density.

CONCLUSIONThe rAAV2 can transfect neurospheres specifically and efficiently. Reporter gene can be expressed in the neurospheres in vivo and ex vivo. Expression of reporter gene can be adjusted by HRE.