NDLI: Transient and selectable transformation of the parasitic protist Trichomonas vaginalis

Content Provider	PubMed Central
Author	Delgadillo, Maria G. Liston, David R. Niazi, Kayvan Johnson, Patricia J.
Copyright Year	1997
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 α-succinyl CoA synthetase B (α-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 μg closed-circular construct. Optimal levels of expression were observed using ≈2.5 × 108 T. vaginalis cells and 350 volts, 960 μFd 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 μg of the NEO construct were plated in the presence of 50 μg/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.
Starting Page	4716
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	9
Volume Number	94
Language	English
Publisher	The National Academy of Sciences of the USA
Publisher Date	1997-04-29
Access Restriction	Open
Rights Holder	The National Academy of Sciences of the USA
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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