Selection based on the expression of antisense hypoxanthine-xanthine-guanine-phosphoribosyltransferase RNA in Toxoplasma gondii.
We have previously shown that an antisense RNA strategy can be used to inhibit the expression of hypoxanthine-xanthine-guanine-phosphoribosyltransferase (HXGPRT) in Toxoplasma gondii [Nakaar et al., J. Biol. Chem. 1999;274:5083-5087]. Here, we report that parasites rendered deficient in HXGPRT by antisense RNA are resistant to high doses of 6-thioxanthine (6-TX). We have exploited this finding to develop a selection procedure. In this scheme, parasites transfected with a chimeric construct harboring the bacterial chloramphenicol acetyl transferase (CAT) reporter gene linked to antisense HXGPRT gene were selected in 6-TX to inhibit the growth of tachyzoites expressing endogenous HXGPRT. Concomitant with a reduction in HXGPRT levels by antisense RNA, 6-TX(R) parasites displayed reporter CAT activity. These data indicate that transfection of antisense HXGPRT gene provides a means to select for parasites expressing foreign or altered genes in T. gondii. These findings also suggest, in principle, that antisense RNA can be used as a strategy to generate selectable markers employing genes that encode enzymes with known subversive substrates.
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Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8022, USA.
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D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D010430:Pentosyltransferases / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014162:Transfection; D014970:Xanthines / Q000494:pharmacology
D000963:Antimetabolites; D016372:RNA, Antisense; D014970:Xanthines; C054700:6-thioxanthine; D015500:Chloramphenicol O-Acetyltransferase; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase