Development and characterization of an inducible RNAi system in Toxoplasma gondii

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.

Name thesis:degree_name

M.Sc.

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Chemistry and Biochemistry

Grantor

University of Windsor

Year dc:date.issued

2009

Author dc:creator

• Vasavan, Biju

Advisor dc:contributor.advisor

• Sirinart Ananvoranich

Contributors dc:contributor

• olsenc@uwindsor.ca

Licence

• http://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

en_CA

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14776/6926

OAI identifier oai:identifier

oai:uwindsor.scholaris.ca:20.500.14776/6926