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PLoS One. 2013 Aug 30;8(8):e73783. doi: 10.1371/journal.pone.0073783. eCollection 2013.

Inducible knockdown of Plasmodium gene expression using the glmS ribozyme.

Author information

1
National Center for Genetic Engineering and Biotechnology (BIOTEC), Khlong Nueng, Khlong Luang, Pathum Thani, Thailand.

Abstract

Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3' untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.

PMID:
24023691
PMCID:
PMC3758297
DOI:
10.1371/journal.pone.0073783
[Indexed for MEDLINE]
Free PMC Article

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