Selection based on the expression of antisense hypoxanthine-xanthine-guanine-phosphoribosyltransferase RNA in...

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.