| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2824115 | Plasmid | 2013 | 7 Pages |
•Development of a NDV infectious clone was a difficult and lengthy process.•A novel and robust ligation-independent cloning (LIC) method was exploited.•A NDV minigenome rescue system was developed within three weeks by LIC.•The minigenome system was fully functional in modeling NDV life cycle.•LIC can be used for rapid development of recombinant vaccines and gene therapy.
Newcastle disease virus (NDV) can cause serious diseases and substantial economic losses to the poultry industry. To gain a better understanding of NDV pathogenesis, several reverse genetics systems for different NDV strains have been established. However, the construction of infectious cDNA clone by conventional restriction digestion/ligation cloning methods is a time-consuming process and has many drawbacks by its nature. To address the problems, we employed a novel and robust ligation-independent cloning (LIC) method for efficient assembly of multiple DNA fragments. Using this method, we successfully generated a NDV minigenome construct within three weeks by assembling RT-PCR products of the VG/GA strain genomic termini and a cDNA coding for the green fluorescence protein (GFP), as a reporter, into a modified pBluescript vector. Co-transfection of the NDV minigenome with three supporting plasmids expressing the N, P, and L proteins into MVA-T7 infected HEp-2 cells and followed by infection with NDV VG/GA resulted in the minigenome replication, transcription, and packaging as evidenced by the reporter gene GFP expression. These results suggest that this LIC approach is a powerful tool for all sequence-independent DNA cloning and multi-DNA fragment assembly, which has a potential application for rapid development of gene therapy and recombinant vaccines.
