title: Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii.
abstract: The obligate intracellular protozoan parasite, Toxoplasma gondii exists as 2 life-cycle forms in intermediate hosts. The rapidly dividing tachyzoites responsible for acute disease, present in the first 14 days of infection, give rise to slowly dividing bradyzoites that reside in tissue cysts. Reactivation of disease is associated with conversion of bradyzoites to tachyzoites. A sensitive method for detection and assessment of the number of each life-cycle stage would be useful for following these events. Herein we describe the construction and validation of a plasmid (pSWITCH) containing a polycompetitor construct (SWITCH) for use in competitive reverse transcriptase-PCR (cRT-PCR). pSWITCH contains competitors for SAG2A and LDH2 genes, which are exclusively expressed by tachyzoite and bradyzoite stages respectively, and for beta-tubulin, a gene expressed by both stages. Using cRT-PCR, samples can first be accurately normalized for expression of the housekeeping gene, beta-tubulin and then the relative levels of SAG2A and LDH2 expression compared to follow stage conversion. The abundance of transcripts for other genes of interest can then be followed during this process as demonstrated here for the SAG2-related family of genes. This technique offers a powerful tool for studying the processes involved in tachyzoite and bradyzoite interconversion.
authors.affiliation: Department of Immunology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, Scotland, UK.
authors.forename: R E
authors.initials: RE
authors.lastname: Lyons
authors.forename: K
authors.initials: K
authors.lastname: Lyons
authors.forename: R
authors.initials: R
authors.lastname: McLeod
authors.forename: C W
authors.initials: CW
authors.lastname: Roberts
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D016054:DNA, Protozoan / Q000737:chemistry; D005786:Gene Expression Regulation; D005812:Genes, Switch / Q000235:genetics*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D016053:RNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014122:Toxoplasma / Q000737:chemistry / Q000235:genetics / Q000254:growth & development*; D014123:Toxoplasmosis / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014404:Tubulin / Q000737:chemistry / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D016054:DNA, Protozoan; D007527:Isoenzymes; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D014404:Tubulin; C067547:surface antigen P22, Toxoplasma; D007770:L-Lactate Dehydrogenase; C409676:lactate dehydrogenase 2