Construction of hNGF-betarecombinant adenovirus and screening of its expression after transfection into various cell lines

ABSTRACT

Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-betarecombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-betagene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-betawhich was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-betarecombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-betarecombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-betarecombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-betarecombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-betagene transfected cells.