ArticleProceedings of the National Academy of Sciences of the United States of America1995
Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1995. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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45 citing papers in PubMed, 148 citations in OpenAlex.
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- Glutamic acid-lysine (EK) rich motif of RabD2 self-associates and regulates adhesion through multivesicular bodies in Entamoeba histolytica.BMC biology · 2025Article
- Work with me here: variations in genome content and emerging genetic tools in Entamoeba histolytica.Trends in parasitology · 2025Review
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- Glycogen Metabolism and Its Role in Growth and Encystation inLife (Basel, Switzerland) · 2023Article
- The Role of Acetate Kinase in the Human ParasiteParasitologia (Basel, Switzerland) · 2022Article
- EhVps23, an ESCRT-I Member, Is a Key Factor in Secretion, Motility, Phagocytosis and Tissue Invasion byFrontiers in cellular and infection microbiology · 2022Article
- Growth and Genetic Manipulation of Entamoeba histolytica.Current protocols · 2022Article
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- Ubc7/Ube2g2 ortholog in Entamoeba histolytica: connection with the plasma membrane and phagocytosis.Parasitology research · 2018Article
- A Calcium/Cation Exchanger Participates in the Programmed Cell Death andFrontiers in cellular and infection microbiology · 2018Article
- TheFrontiers in cellular and infection microbiology · 2018Article
- Structure of Ca2+-binding protein-6 from Entamoeba histolytica and its involvement in trophozoite proliferation regulation.PLoS pathogens · 2017Article
- The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation inThe Journal of biological chemistry · 2017Article
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- InMicrobial cell (Graz, Austria) · 2015Article
- EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica.PLoS pathogens · 2015Article
- Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.
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