title: Transient and selectable transformation of the parasitic protist Trichomonas vaginalis.
abstract: We have developed methods to transiently and selectably transform the human-infective protist Trichomonas vaginalis. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into T. vaginalis by electroporation and have used the 5' and 3' untranslated regions of the endogenous gene alpha-succinyl CoA synthetase B (alpha-SCSB) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 microg closed-circular construct. Optimal levels of expression were observed using approximately 2.5 x 10(8) T. vaginalis cells and 350 volts, 960 microFd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in T. vaginalis transient transformants revealed the highest level of expression 8-21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (neo) gene as the selectable marker. Cells electroporated with 20 microg of the NEO construct were plated in the presence of 50 microg/ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3-5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10-30 copies per cell. The ability to transiently and selectably transform T. vaginalis should greatly enhance research on this important human parasite.
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Delgadillo
authors.forename: D R
authors.initials: DR
authors.lastname: Liston
authors.forename: K
authors.initials: K
authors.lastname: Niazi
authors.forename: P J
authors.initials: PJ
authors.lastname: Johnson
mesh_terms: D000818:Animals; D000994:Antitrichomonal Agents / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004351:Drug Resistance; D018274:Electroporation; D017930:Genes, Reporter; D005819:Genetic Markers; D019868:Kanamycin Kinase; D008156:Luciferases / Q000235:genetics; D010303:Paromomycin / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012641:Selection, Genetic; D014170:Transformation, Genetic; D014246:Trichomonas vaginalis / Q000235:genetics*
chemical_list: D000994:Antitrichomonal Agents; D005819:Genetic Markers; D010303:Paromomycin; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase