A toolbox facilitating stable transfection of Eimeria species.

Molecular and Biochemical Parasitology
Julie ClarkDamer P Blake

Abstract

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isol...Continue Reading

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Citations

Feb 2, 2013·Parasitology Research·Guangwen YinXun Suo
Oct 31, 2012·Journal of Biosciences·Emily L Clark, Damer P Blake
Jul 19, 2013·Journal of Parasitology Research·Hong ChenMichael Kurth
Nov 19, 2013·Trends in Parasitology·Damer P Blake, Fiona M Tomley
Sep 14, 2014·Parasitology Research·Na ZhaoXichen Zhang
Mar 8, 2014·Poultry Science·H D Chapman
Oct 19, 2010·Avian Pathology : Journal of the W.V.P.A·Pete Kaiser
Apr 22, 2009·International Journal for Parasitology·Jun ZouXun Suo
May 20, 2015·Veterinary Parasitology·Damer P Blake
Aug 17, 2014·Biochemical and Biophysical Research Communications·Na ZhaoXichen Zhang
Sep 25, 2012·International Journal for Parasitology·Joachim Müller, Andrew Hemphill
Jul 28, 2009·Molecular and Biochemical Parasitology·Wei LiXichen Zhang
Aug 25, 2016·Parasites & Vectors·Virginia Marugan-HernandezFiona M Tomley
Feb 28, 2019·Current Protocols in Microbiology·Iván Pastor-FernándezVirginia Marugán-Hernández
Feb 26, 2020·Infection and Immunity·Xinming TangXun Suo
Apr 4, 2019·Cellular Microbiology·Mamadou Amadou DialloAnne Silvestre
Jul 22, 2017·Frontiers in Cellular and Infection Microbiology·Bingjian Ren, Nishith Gupta
Oct 19, 2017·Parasites & Vectors·Virginia Marugan-HernandezFiona M Tomley
May 16, 2018·Antimicrobial Agents and Chemotherapy·Hong-Bin YanGabriel Rinaldi
Mar 12, 2020·Frontiers in Bioengineering and Biotechnology·Dandan HuXianyong Liu
Oct 12, 2021·Frontiers in Genetics·Peipei ChengFeng Gu
Jul 13, 2014·Genome Research·Adam J ReidArnab Pain

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