Dynamic development of parasitophorous vacuole of Eimeria tenella transfected with the yellow fluorescent protein gene fused to different signal sequences from apicomplexan parasites

Shi, Tuanyuan; Yan, Wenchao; Ren, Huaibin; Liu, Xianyong; Suo, Xun

doi:10.1007/s00436-008-1194-y

Cited by 13 publications

(6 citation statements)

References 17 publications

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“…1B) was constructed by cutting the fragment containing the EYFP sequence under the control of the E. tenella Histone 4 promoter and incorporating a 5′ 90-bp nuclear location signal sequence (NLS) and a 3′ E. tenella actin 3′ UTR from pH-2E-A3′ [26] using Eco RV and Bgl II. The digestion product was cloned between the ITRs of the piggyBac element in the vector pXL-BAC II [25].…”

Section: Methodsmentioning

confidence: 99%

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

Liu

Yan

et al. 2012

PLoS ONE

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piggyBac , a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella . Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5′ and 3′ ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac .

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Section: Methodsmentioning

confidence: 99%

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

Liu

Yan

et al. 2012

PLoS ONE

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“…Briefly, the EYFP gene of pHIS-EYFP/ACT-RFP was replaced by DHFR-EYFP gene following the RFP gene was replaced by ssChIL- 2 gene amplified by three round PCR by the use of the primers ChIL-2-R and ChIL-2-1, ChIL-2-2 and ChIL-2-3, respectively. A signal sequence (ss) was obtained from T. gondii GRA 8, which functionally regulates ChIL-2 secretion ( Shi et al, 2009 ; Zou et al, 2009 ; Yin et al, 2011 ). The plasmid DNA was linearized by the SnaB I restriction enzyme, which released the two expression cassettes from the backbone of the plasmid ( Figure 1A ).…”

Section: Methodsmentioning

confidence: 99%

Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites

Tang

Suo

et al. 2015

Front. Microbiol.

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Chicken coccidiosis, caused by Eimeria sp., occurs in almost all poultry farms and causes huge economic losses in the poultry industry. Although this disease could be controlled by vaccination, the reduced feed conservation ratio limits the widespread application of anticoccidial vaccines in broilers because some intermediate and/or low immunogenic Eimeria sp. only elicit partial protection. It is of importance to enhance the immunogenicity of these Eimeria sp. by adjuvants for more effective prevention of coccidiosis. Cytokines have remarkable effects on the immunogenicity of antigens. Interleukin 2 (IL-2), for example, significantly stimulates the activation of CD8+ T cells and other immune cells. In this study, we constructed a transgenic Eimeria mitis line (EmiChIL-2) expressing chicken IL-2 (ChIL-2) to investigate the adjuvant effect of ChIL-2 to enhance the immunogenicity of E. mitis against its infection. Stable transfected EmiChIL-2 population was obtained by pyrimethamine selection and verified by PCR, genome walking, western blotting and indirect immunofluorescence assay. Cellular immune response, E. mitis-specific IFN-γ secretion lymphocytes in the peripheral blood mononuclear cells, stimulated by EmiChIL-2 was analyzed by enzyme-linked immunospot assay (ELISPOT). The results showed that EmiChIL-2 stimulated a higher cellular immune response compared with that of the wild-type parasite infection in chickens. Moreover, after the immunization with EmiChIL-2, elevated cellular immune response as well as reduced oocyst output were observed These results indicated that ChIL-2 expressed by Eimeria sp. functions as adjuvant and IL-2 expressing Eimeria parasites are valuable vaccine strains against coccidiosis.

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“…1). For example, the reporter enhanced yellow fluorescent protein (EYFP) fused with the signal sequence of dense granule antigen 8 (GRA8) of T. gondii was mainly secreted to the parasitophorous vacuole (PV) in Eimeria tenella controlled by the Actin promoter (25,31).…”

Section: Transient-transfection Systemmentioning

confidence: 99%

Towards Innovative Design and Application of Recombinant Eimeria as a Vaccine Vector

2020

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Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

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Dynamic development of parasitophorous vacuole of Eimeria tenella transfected with the yellow fluorescent protein gene fused to different signal sequences from apicomplexan parasites

Cited by 13 publications

References 17 publications

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

piggyBac Transposon-Mediated Transgenesis in the Apicomplexan Parasite Eimeria tenella

Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites

Towards Innovative Design and Application of Recombinant Eimeria as a Vaccine Vector

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