[Subcloning of human neurotrophin-3 gene and construction of its genetically engineered cell model]

To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro.

Human BM-MSCs were cultured in low-glucose DMEM supplemented with 10% fetal bovine serum and 10 ng/ml epidermal growth factor. Flow cytometry (FCM) was used to examine the phenotypes of the cells. The eukaryotic expression vector pcDNA3.1(+)/NT3 was constructed and transferred into human BM-MSCs in vitro via liposomes. The genetically engineered BM-MSCs were selected several times with G418 and the clones were obtained and then amplified, followed by extraction of the RNA for detection of NT3 gene expression by reverse transcriptional (RT) PCR. The biological activity of the genetically engineered cells was examined by the collecting the supernatant of the culture medium for incubation of guinea pig cochlea hair cells.

The cultured cells expressed CD13, CD29 and CD59, but no7 CD11, CD14, CD31, CD34, CD45, CD80, CD86, CD117 or HLA-DR. The BM-MSCs genetically modified with pcDNA3.1(+)/NT3 not only expressed and produced NT3, but also promoted the survival of the guinea pig cochlea hair cells in vitro.

It is possible to construct the genetically engineered BM-MSCs that excrete NT3 in vitro.