title: Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo.
abstract: Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil.
authors.forename: T C
authors.initials: TC
authors.lastname: Paredes-Santos
authors.affiliation: Departments of Pathology and Medicine, Albert Einstein College of Medicine - AECOM, USA.
authors.forename: T
authors.initials: T
authors.lastname: Tomita
authors.affiliation: Departments of Pathology and Medicine, Albert Einstein College of Medicine - AECOM, USA.
authors.forename: M
authors.initials: M
authors.lastname: Yan Fen
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil.
authors.forename: W
authors.initials: W
authors.lastname: de Souza
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil.
authors.forename: M
authors.initials: M
authors.lastname: Attias
authors.affiliation: Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil. Electronic address: vommaro@biof.ufrj.br.
authors.forename: R C
authors.initials: RC
authors.lastname: Vommaro
authors.affiliation: Departments of Pathology and Medicine, Albert Einstein College of Medicine - AECOM, USA. Electronic address: louis.weiss@einstein.yu.edu.
authors.forename: L M
authors.initials: LM
authors.lastname: Weiss
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D018929:Cell Culture Techniques; D002460:Cell Line; D004847:Epithelial Cells / Q000469:parasitology*; D005347:Fibroblasts / Q000469:parasitology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D011401:Promoter Regions, Genetic; D013194:Staining and Labeling / Q000379:methods*; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development*; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Atypical strain; Bradyzoite; Conversion; Cyst; Toxoplasma gondii