| Toxoplasma gondii is a ubiquitous protozoan parasite that can infect and replicate within any nucleated mammalian or avian cell. The ability of double stranded RNA to downregulate gene expression has been demonstrated in T. gondii. We have constructed a tetracycline based inducible RNAi vector which can be used for regulating gene expression in T. gondii. Upon the addition of anhydrotetracycline, the production of double stranded RNA homologous to the target gene is induced. The inducible vector was characterized using the marker gene, uracil phosphoribosyl transferase. The inducible vector was stably transfected into RHtetR strain of the parasite and specifically lowered the expression of uracil phosphoribosyl transferase detected by RT-PCR, uracil incorporation assay and 5-fluro-2'-deoxyuridine resistance. Subsequently, the inducible RNAi vector was used to elucidate the role of microneme protein 3 (MIC 3), which is an invasion related gene. We generated a stable transgenic parasite line in which the expression of MIC 3 was knocked down detected by RT-PCR. However, we did not detect any changes in the growth of the parasite or its invasion ability, as monitored by nucleotide uptake assay and growth assay. |
