A toolbox facilitating stable transfection of Eimeria species

Clark, Julie D.; Billington, Karen; Bumstead, Janene M.; Oakes, Richard D.; Soon, Puay Eng; Sopp, P.; Tomley, Fiona M.; Blake, Damer P.

doi:10.1016/j.molbiopara.2008.07.006

Cited by 62 publications

(100 citation statements)

References 26 publications

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“…Direct transfection of trichomonad cells was attempted using cytomix as the electroporation buffer previously described for Eimeria transfection (Clark et al 2008). Five cuvettes (BTX, 0.4 cm) numbered 1 to 5 were prepared for transfection and filled with 300 μl (1×10 7 ) of cell suspension each.…”

Section: Parasite Transfection and G418 Selectionmentioning

confidence: 99%

The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis

Zhang

et al. 2011

Parasitol Res

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Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis.

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Section: Parasite Transfection and G418 Selectionmentioning

confidence: 99%

The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis

Zhang

et al. 2011

Parasitol Res

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“…Approximately 459 bp and 293 bp of 5' and 3' untranslated region (UTR) of CPV-S were respectively amplified by PCR using pCPVS plasmid as templates and primer pairs S 1 F/S 1 R and S 2 F/S 2 R (Table 1B). The GFP coding gene was amplified from the pGFP-C1 plasmid by PCR with primers GFP-F and GFP-R (Table 1B) Cryptosporidium was transfected using cytomix as the electroporation buffer previously described for Eimeria transfection [41]. Freshly purified oocysts and excysted sporozoites were washed twice in incomplete cytomix (10 mM K 2 HPO 4 /KH 2 PO 4 , pH 7.6, 120 mM KCl, 0.15 mM CaCl 2 , 25 mM HEPES, 2 mM EGTA and 5 mM MgCl 2 ).…”

Section: Plasmid Constructionsmentioning

confidence: 99%

“…High transfection efficiency has been achieved for T. gondii, which is easily cultured in vitro and as a result, has been utilized as a versatile genetic model organism to study the basic biology of other Apicomplexan parasites [36,37]. Successful transfection of Eimeria was achieved using a series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that conferred resistance to the drug pyrimethamine [41]. They are highly specific to their host and (3).…”

Section: Transient Expression Of Gfp In Transfected C Parvum Oocystsmentioning

confidence: 99%

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker

Zhang

Liang

et al. 2009

Molecular and Biochemical Parasitology

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…Oocysts were propagated as described elsewhere (Clark et al 2008). Sporozoites and second generation merozoites were purified following standard protocols (Shirley 1995).…”

Section: E Tenella Propagation and Purificationmentioning

confidence: 99%

“…Additional negative control primers were designed from the sequence previously described as the putative signal peptide . PCR amplification, PCR product cloning and sequencing were all performed as described previously (Clark et al 2008).…”

Section: Reverse Transcription-polymerase Chain Reactionmentioning

confidence: 99%

Eimeria tenella microneme protein EtMIC4: capture of the full-length transcribed sequence and comparison with other microneme proteins

et al. 2008

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EtMIC4 is a microneme protein of Eimeria tenella, an intracellular apicomplexan protozoan that can cause severe enteritis in chickens. The EtMIC4 gene has been partially characterised, and in this study, we used a combined strategy of rapid amplification of cDNA ends (5'RACE) and reverse transcription-polymerase chain reaction to identify the authentic 5' end of the transcribed sequence (accession number AJ306453.2). Comparison of the predicted EtMIC4 transcription start site with predicted start sites for EtMIC1, 2 and 3 genes identified comparable initiator regions that each conform to the consensus sequence for a transcriptional initiator element. The EtMIC4 gene is organised over 11 exons and analysis of the full-length predicted protein identified a new N-terminal region that comprises a hydrophobic signal peptide followed by four thrombospondin-type 1 modules that are similar to those previously described further downstream in the protein. Best-fit analysis shows that EtMIC4 shares high homology with the Eimeria maxima protein EmTFP250 and with TgMIC12, a predicted Toxoplasma gondii microneme protein. EtMIC4 and EmTFP250 share 70% amino acid identity and all predicted structural domains are conserved between the two. EtMIC4 and TgMIC12 share 48% identity and they have very similar domain organisation and conservation of intron/exon boundaries.

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A toolbox facilitating stable transfection of Eimeria species

Cited by 62 publications

References 26 publications

The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis

The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker

Eimeria tenella microneme protein EtMIC4: capture of the full-length transcribed sequence and comparison with other microneme proteins

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