Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.
Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.
Arii
Jun
J
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Hushur
Orkash
O
Kato
Kentaro
K
Kawaguchi
Yasushi
Y
Tohya
Yukinobu
Y
Akashi
Hiroomi
H
D000818:Animals; D001426:Bacterial Proteins; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral; D002184:Herpesvirus 1, Canid / Q000235:genetics* / Q000254:growth & development; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection
D001426:Bacterial Proteins; C113731:Flp protein, bacteria; D013937:Thymidine Kinase