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 inhi bit the expression of hypoxanthine-xanthine-guanine-phosphoribosyltransfera se (HXGPRT) in Toxoplasma gondii [Nakaar et al., J. Biol. Chem. 1999;274:50 83-5087]. Here, we report that parasites rendered deficient in HXGPRT by an tisense RNA are resistant to high doses of 6-thioxanthine (6-TX). We have e xploited this finding to develop a selection procedure. In this scheme, par asites transfected with a chimeric construct harboring the bacterial chlora mphenicol 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 antise nse RNA, 6-TXR parasites displayed reporter CAT activity. These data indica te that transfection of antisense HXGPRT gene provides a means to select fo r parasites expressing foreign or altered genes in T. gondii. These finding s also suggest, in principle, that antisense RNA can be used as a strategy to generate selectable markers employing genes that encode enzymes with kno wn subversive substrates. (C) 2000 Elsevier Science B.V. All rights reserve d.