Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine.
The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.
Holzer
Georg W
GW
Baxter BioScience/Vaccines, Biomedical Research Center, Uferstrasse 15, A-2304 Orth/Donau, Austria.
Mayrhofer
Josef
J
Gritschenberger
Werner
W
Falkner
Falko G
FG
D000818:Animals; D019380:Anti-HIV Agents / Q000378:metabolism; D019677:Artificial Gene Fusion; D001711:Biotransformation; D002460:Cell Line; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics*; D011355:Prodrugs; D013937:Thymidine Kinase / Q000235:genetics*; D014616:Vaccinia virus / Q000201:enzymology*; D015215:Zidovudine / Q000378:metabolism*
D019380:Anti-HIV Agents; D011355:Prodrugs; D015215:Zidovudine; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase