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Application of the ligation-independent cloning (LIC) method

Author:
Zhao, Wei, Hu, Haixia, Zsak, Laszlo, Yu, Qingzhong, Yang, Zengqi
Source:
Plasmid 2013 v.70 no.3 pp. 314
ISSN:
0147-619X
Subject:
Newcastle disease virus, complementary DNA, gene therapy, green fluorescent protein, human cell lines, molecular cloning, pathogenesis, plasmids, recombinant vaccines, reporter genes, reverse transcriptase polymerase chain reaction
Abstract:
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.
Handle:
10113/59840