Construction and identification of recombinant retroviral vector carrying NGF gene

Objective To construct recombinant retroviral vector carrying rat NGF gene, and to observe its expression in neural stem cell (NSC). Methods Total RNA was extracted from rat brain hippocampus as the template and the β,NGF gene was amplified by reverse transcription,polymerase chain reaction (RT,PCR). By gene recombination technique, rat β,NGF cDNA was inserted into retroviral vector pLEGFP,N1. And then, we transfected the recombinant plasmid into packaging cell PT67 by Lipofectamine 2000, and collected the virus supernatant of positive clones after G418 selection and used it to infect NSC. After that, we observed the neurite outgrowth of PC12 cell stimulated by NGF expressed by NSC. Results The recombinant plasmid was identified by restriction endonuclease analysis. The β,NGF gene could be expressed successfully by NSC. The culture supernatant of NSC could stimulate the neurite outgrowth of PC12 cell. Conclusion The recombinant retroviral vector pLEGFP,NGF was constructed successfully. The β,NGF gene could be expressed successfully by NSC and with good biological activity.