Trypanosoma cruzi: use of herpes simplex virus-thymidine kinase as a negative selectable marker.
Trypanosoma cruzi, the protozoan that causes Chagas' disease, was transfected with a fusion gene of hygromycin phosphotransferase and herpes simplex virus-thymidine kinase, HyTK. Transfectants selected in hygromycin had thymidine kinase activity, whereas controls did not. In vitro growth of the mammalian life-stage forms, amastigotes and trypomastigotes, was inhibited 98% by the nucleoside analogue ganciclovir (5 micrograms/ml). Growth of the insect-stage form, epimastigotes, was not inhibited by ganciclovir (up to 250 micrograms/ml) or other nucleoside analogues. Intracellular uptake of ganciclovir by epimastigotes was found to be 10-fold less than that by amastigotes. Mice infected with the HyTK-expressing parasites and treated with ganciclovir had a statistically significant reduction of parasitemia by 57%; however, complete eradication of parasites was not achieved. The parasites recovered from the treated mice continued to be susceptible to ganciclovir in vitro. Parasite clones with higher expression of thymidine kinase were more sensitive to ganciclovir, suggesting that greater expression of the thymidine kinase gene may lead to parasites that can be fully eradicated from infected experimental animals.
Buckner
F S
FS
Department of Medicine, University of Washington, Seattle 98195, USA.
Wilson
A J
AJ
Van Voorhis
W C
WC
D016475:3T3 Cells; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D015139:Blotting, Southern; D014355:Chagas Disease / Q000188:drug therapy; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D015774:Ganciclovir / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D015971:Gene Expression Regulation, Enzymologic; D005819:Genetic Markers; D051379:Mice; D018512:Parasitemia / Q000188:drug therapy; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
D000998:Antiviral Agents; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D013937:Thymidine Kinase; D015774:Ganciclovir; D013936:Thymidine