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

Valerian Nakaar, Emily O. Ngo, Keith A. Joiner

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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. (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)43-51
Number of pages9
JournalMolecular and Biochemical Parasitology
Volume110
Issue number1
DOIs
StatePublished - Sep 1 2000

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Keywords

  • Antisense RNA
  • HXGPRT
  • Selection
  • Toxoplasma

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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