The Establishment of GFP Stably Expressing Cell Line Using Replication Incompetent Lentiviral Vectors

Objectives:To establish a green fluorescent protein (GFP) stably expressing cell line using replication incompetent lentiviral vector. Methods: Vector plasmid pHR'-pCMV-GFP containing GFP gene as a target gene, package construct pCMV- ΔR8.2 and an envelope plasmid pCMV-VSVG were co-transfected into 293T packaging cell line using Ca-P precipitation method. The viruses harvested from the supernatant were used to transfect 293T cells again. Then the GFP strongly expressing monoclone was selected with a clone ring and then passaged. After 12 passages, the cells were sent for FACS to convince the stability of the GFP expression. Besides, a GFP targeted RNA interference experiment was conducted to assure that this cell line could be used in gene expression regulation experiments. Results: A GFP stably expressing cell line G4 was established, in this cell line more than 99% of the cells expressed GFP, which was stable after at least 12 passages and could be efficiently downregulated by RNAi. Conclusion: These researches showed that the lentiviral vector was efficient to establish GFP expressing cell lines, and using this vector a GFP stably expressing cell line G4 was established, which could be used efficiently in gene regulation research.