Application of the ligation-independent cloning (LIC) method for rapid construction of a minigenome rescue system for Newcastle disease virus VG/GA strain.
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.
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Southeast Poultry Research Laboratory, Agricultural Research Services, United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA.
Wei
W
Zhao
Haixia
H
Hu
Laszlo
L
Zsak
Qingzhong
Q
Yu
Zengqi
Z
Yang
D045744:Cell Line, Tumor; D022201:Chromosomes, Artificial; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004847:Epithelial Cells / Q000821:virology; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009522:Newcastle disease virus / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012367:RNA, Viral; D049452:Green Fluorescent Proteins
Green fluorescence protein (GFP) expression; Ligation-independent cloning (LIC); Minigenome rescue; NDV VG/GA strain