[Construction of recombinant adenovirus vector pAdxsi-green fluorescent protein-homo sapiens NEL-like 1 and transfected into rat bone marrow mesenchymal stem cells]

Jing Xue, Jiang Peng, Li Zhang, Shuyun Liu, Jifeng Chen, Aiyuan Wang, Mei Yuan, Wenjing Xu, Shibi Lu
Chinese Journal of Reparative and Reconstructive Surgery 2010, 24 (5): 606-12

OBJECTIVE: To construct a recombinant adenovirus vector pAdxsi-GFP-NELL1 that co-expressing green fluorescent protein (GFP) and homo sapiens NEL-like 1 (NELL1) protein (a protein strongly expressed in neural tissue encoding epidermal growth factor like domain), to observe its expression by transfecting the recombinant adenovirus into rat bone marrow mesenchymal stem cells (BMSCs) so as to lay a foundation for further study on osteogenesis of NELL1 protein.

METHODS: From pcDNA3.1-NELL1, NELL1 gene sequence was obtained, then NELL1 gene was subcloned into pShuttle-GFP-CMV (-)TEMP vector which was subsequently digested with enzyme and inserted into pAdxsi vector to package the recombinant adenovirus vector (pAdxsi-GFP-NELL1). After verified by enzyme cutting and gel electrophoresis, pAdxsi-GFP-NELL1 was amplified in HEK293 cells and purified by CsCl2 gradient purification, titrated using 50% tissue culture infective dose (TCID50) assay. The rat BMSCs were cultured and identified by flow cytometry and directional induction, then were infected with adenoviruses (pAdxsi-GFP-NELL1 and pAdxsi-GFP). NELL1 expression was verified by RT-PCR and immunofluorescence; GFP gene expression was verified by the intensity of green fluorescence under fluorescence microscope. Cell counting kit-8 (CCK-8) was used for investigate the influence of vectors on the proliferation of rat BMSCs.

RESULTS: Recombinant adenoviral vector pAdxsi-GFP-NELL1, which encodes a fusion protein of human NELL1, was successfully constructed and amplified with titer of 1 x 10(11) pfu/mL. The primary BMSCs were cultured and identified by flow cytometric analysis, osteogenic and adipogenic induction, then were used for adenoviral transfection efficiency and cell toxicity tests. An multiplicity of infection of 200 pfu/cell produced optimal effects in transfer efficiency without excessive cell death in vitro. Three days after transfection with 200 pfu/cell pAdxsi-GFP-NELL1 or pAdxsi-GFP, over 60% BMSCs showed green fluorescent by fluorescence microscopy. Immunofluorescence with NELL1 antibody also revealed high level expression of human NELL1 protein in red fluorescent in these GFP expressing cells. RT-PCR analysis confirmed that the exogenous expression of NELL1 upon transfection with pAdxsi-GFP-NELL1 at 200 pfu/cell, whereas NELL1 remained undetectable in Ad-GFP-transfected rat BMSCs. The proliferative property of primary rat BMSCs after adenoviral NELL1 transfection was assayed by CCK-8 in growth medium. Growth curve demonstrated no significant difference among BMSCs transfected with pAdxsi-GFP-NELL1, pAdxsi-GFP, and no treatment control at 7 days (P > 0.05).

CONCLUSION: Recombinant adenovirus vector pAdxsi-GFP-NELL1 can steady expressing both GFP and NELL1 protein after being transfected into rat BMSCs. It provides a useful tool to trace the expression of NELL1 and investigate its function in vitro and in vivo.

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