InFusion Cloning for the Generation of Biologically Relevant HCV Chimeric Molecular Clones
This chapter describes how to generate chimeric molecular cassettes that are ready to receive PCR-amplified E1/E2 genes using new DNA cloning technology. The method is divided into three sections: (1) generation of a ΔCore-NS2 cassette based upon the full-length JFH-1 molecular clone; (2) insertion of a “structural gene” fragment encoding the Core, p7, and NS2 genes of a given genotype reference sequence, to generate a ΔE1/E2 cassette; and (3) insertion of patient-isolated E1/E2 genes that are genotype-matched to the structural genes. The final assembled chimeric genomes can then be analyzed in the HCV cell culture system. These cassettes allow characterization of the extensive in vivo viral diversity without the need to isolate and clone whole virus genomes. This method can be readily applied to the study of other HCV genes and other viruses.
Key wordsHepatitis C virus Chimeric virus Full-length molecular clones Patient-isolated E1/E2
The authors would like to thank Takaji Wakita for plasmid pJFH-1 and Chris Lounds for molecular biology discussions. This work was supported by the Medical Research Council UK (G0801169) and by the EU FP7 Grant “HepaMAb” (305600).