title: Generation of Recombinant Capripoxvirus Vectors for Vaccines and Gene Knockout Function Studies.
abstract: The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is used to generate capripoxvirus knockout viruses (KO), and is based on the targeting a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses.
authors.affiliation: Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3M4.
authors.forename: Hani
authors.initials: H
authors.lastname: Boshra
authors.affiliation: Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3R2.
authors.forename: Jingxin
authors.initials: J
authors.lastname: Cao
authors.affiliation: Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, MB, Canada, R3E 3M4. Shawn.babiuk@inspection.gc.ca.
authors.forename: Shawn
authors.initials: S
authors.lastname: Babiuk
mesh_terms: D018151:Capripoxvirus / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014614:Vaccines, Synthetic / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use
chemical_list: D004274:DNA, Recombinant; D014614:Vaccines, Synthetic; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Capripoxvirus; E. coli gpt selection; EGFP; Recombinant virus; Virus titration