Neurturin gene cloning and expression in Vero cells

摘要

BACKGROUND: Transplantation of cell lines expressing neurturin (NTN) has been used to treat animal models of Parkinson's disease. However, gene homology between humans and rats varies greatly, so experimental results are not entirely suitable for understanding cellular transplantation in humans.OBJECTIVE: To explore expression of NTN in African green monkey kidney cells (Veto celts); to obtain a stably NTN expressing cell line.DESIGN, TIME AND SETTING: An observation study of gene engineering and cellular biology was performed at the Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College between 2005 and 2008.MATERIALS: Human embryonic hepatic tissues, expression vector pcDNA3, and Vero cells were prepared in this laboratory. Primers were synthesized by TaKaRa Biotechnology, Dalian, China; RNA extraction kit and plasmid extraction kit were purchased from Shanghai Watson Bioengineering, China; G418 and M'IT were purchased through Sigma, USA; Lipofectamine2000 was a product of Invitrogen, USA; mice anti-human NTN antibody and fluorescent labeling goat anti-mouse IgG antibody were provided by Jingmei Biotech, China.METHODS: Total RNA was harvested from human embryonic hepatic tissues, and NTN cDNA was cloned by RT-PCR method, followed by subcloning into the pcDNA3 eukaryotic expression vector. The obtained pcDNA3/hNTN was stably transfected into Vero cells using Lipofectamine 2000, and stably expressing clones were selected using G418.MAIN OUTCOME MEASURES: NTN mRNA and protein expressions were respectively identified by RT-PCR and immunofluorescence. The morphology of transfected cells was observed under inverted microscopy, and the growth characteristics of those cells were determined using MTT method.RESULTS: A clonal cell line, stably expressing human NTN mRNA and protein, was obtained through stable transfection of pcDNA3/hNTN into Veto cells. The transfected Vero cells exhibited irregular morphology, rather than a spindle shape. The growth retardation phase was prolonged, but the number of cells was identical to non-transfected cells.CONCLUSION: Vero cell lines, which stably expressed human NTN protein, were obtained, and expression patterns of these cell lines were acceptable.