Document Detail


Development of a negative selectable marker for Entamoeba histolytica.
MedLine Citation:
PMID:  21189470     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.
Authors:
Mayuresh M Abhyankar; Sarah M Haviland; Carol A Gilchrist; William A Petri
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Video-Audio Media     Date:  2010-12-12
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2010  
Date Detail:
Created Date:  2010-12-29     Completed Date:  2011-01-20     Revised Date:  2013-07-03    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  -     Citation Subset:  IM    
Affiliation:
Division of Infectious Disease and International Health, University of Virginia Health System, USA.
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MeSH Terms
Descriptor/Qualifier:
Cytosine Deaminase / biosynthesis*,  genetics
Entamoeba histolytica / drug effects*,  enzymology*,  genetics
Flucytosine / pharmacokinetics,  pharmacology*
Pentosyltransferases / biosynthesis*,  genetics
Transfection / methods
Transgenes
Grant Support
ID/Acronym/Agency:
AI 26649/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
2022-85-7/Flucytosine; EC 2.4.2.-/Pentosyltransferases; EC 2.4.2.9/uracil phosphoribosyltransferase; EC 3.5.4.1/Cytosine Deaminase
Comments/Corrections

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