Restriction enzyme-mediated transfection improved transfection efficiency in vitro in Apicomplexan parasite Eimeria tenella

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

doi:10.1016/j.molbiopara.2008.06.006

Cited by 34 publications

(35 citation statements)

References 15 publications

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“…Liu et al (15) achieved 0.2% transfection rate with E. tenella . Later, Qin et al (7) showed a similar rate (0.19%) for E. mitis .…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid used in the transfection, p HDEp2aRA, is a single expression cassette where DHFR-Ts2m3m, a pyrimethamine resistance gene from T. gondii , EYFP, and RFP were inserted between histone 4 promoter with its nuclear localization signal (90 bp) (15) and 3′ untranslated region of actin of E. tenella . A porcine teschovirus-1 2A peptide (P2A) (66 bp), which was shown to be able to cleave two contiguous proteins (10, 16), was added between EYFP and RFP.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, 2 × 10 7 purified sporozoites were electroporated using a nucleofector (Program U-033, AMAXA, Switzerland) by a restriction enzyme-mediated method in a system containing 100 µl cytomix buffer, 15 µg linearized DNA, and 250 UI Sna BI restriction enzyme (15). Sporozoites were resuspended in DMEM and inoculated to MDBK culture for in vitro transient transfection or injected into duodenum of a 4-week-old rabbit in a laparotomy for in vivo stable transfection.…”

Section: Methodsmentioning

confidence: 99%

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Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

et al. 2017

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Rabbit coccidiosis causes great economic losses to world rabbitries. Little work has been done considering genetic manipulation on the etiological agents, rabbit Eimeria spp. In this study, we constructed a transgenic line of Eimeria magna (EmagER) expressing enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP) using regulatory sequences of Eimeria tenella and Toxoplasma gondii. We observed the life cycle of EmagER and confirmed that the transgenic parasites express exogenous proteins targeted to different cellular compartments throughout the entire life cycle. EYFP was expressed mainly in the nucleus and RFP both in the nucleus and cytoplasm. Then, coccidia-free, laboratory-reared 40-day-old rabbits were primarily infected with either EmagER or wild-type strain oocysts and challenged with the wild-type strain. EmagER showed similar reproductivity and immunogenicity to the wild-type strain. Finally, we examined the foreign protein-specific immune response elicited by EmagER. Rabbits were immunized with either transgenic or wild-type oocysts. Immune response against parasite-soluble antigen, EYFP and RFP in spleen, and mesenteric lymph nodes were detected by quantitative real-time PCR. The relative expression level of IFN-γ, IL-2, and TNF-α were higher in EmagER-immunized rabbits than wild-type parasites-immunized rabbits after stimulation with EYFP and RFP. Our study confirmed that a specific immune response was induced by the exogenous protein expressed by EmagER and favored future studies on application of transgenic rabbit coccidia as recombinant vaccine vectors.

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“…Liu et al (15) achieved 0.2% transfection rate with E. tenella . Later, Qin et al (7) showed a similar rate (0.19%) for E. mitis .…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

et al. 2017

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“…Restriction enzyme-mediated integration (REMI) was applied to increase transfection efficiency [9, 22]. For transfection, four wells were used, each containing 1 × 10 6 sporozoites in 20 μl P3 buffer (Lonza), 12 μg of plasmid DNA (in 2 μl) and 6 U Psi I.…”

Section: Methodsmentioning

confidence: 99%

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

et al. 2016

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BackgroundEimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens.ResultsIn this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens.ConclusionsE. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users.

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“…The plasmids were transfected to E. tenella sporozoites by restriction enzyme-mediated integration (16). The plasmid DNA (30 mg) was linearized with BamHI, and the enzyme was removed by phenol extraction.…”

Section: Transfection and Selection Of Transgenic Parasitesmentioning

confidence: 99%

Transgenic Eimeria tenella Expressing Enhanced Yellow Fluorescent Protein Targeted to Different Cellular Compartments Stimulated Dichotomic Immune Responses in Chickens

Huang

Zou

et al. 2011

The Journal of Immunology

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Eimeria tenella, one of the seven species of chicken coccidia, elicits protective immunity against challenge infection with both homologous and heterologous strains. We endeavor to use recombinant E. tenella as a vaccine vehicle for expressing and delivering pathogen Ags and investigate immune responses against these foreign Ags. In this study, two lines of transgenic E. tenella expressing a model Ag, enhanced yellow fluorescent protein (EYFP), targeted to the micronemes and to the cytoplasm of the recombinant parasites were constructed to study the impact of Ag compartmentalization on immunogenicity. The MTT assay, intracellular cytokine staining, and real-time PCR were performed to detect the EYFP-specific proliferation and effector functions of splenic lymphocytes of immunized chickens. ELISA was used to measure anti-EYFP IgG and IgA responses. The results showed that both lines of transgenic parasites stimulated EYFP-specific lymphocyte proliferation and IFN-γ expression in CD4 and CD8 T cells, whereas a higher level of Ag-specific lymphocyte proliferation was elicited by the transgenic line expressing microneme-targeted EYFP. Furthermore, this line stimulated stronger IgA response than the one expressing cytoplasm-targeted EYFP after the second immunization. Our findings are encouraging for further investigation of the effect of Ag compartmentalization in transgenic Eimeria on immunogenicity and for the development of a eukaryotic vaccine vector using genetically modified Apicomplexa parasites.

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Restriction enzyme-mediated transfection improved transfection efficiency in vitro in Apicomplexan parasite Eimeria tenella

Cited by 34 publications

References 15 publications

Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

Transgenic Eimeria magna Pérard, 1925 Displays Similar Parasitological Properties to the Wild-type Strain and Induces an Exogenous Protein-Specific Immune Response in Rabbits (Oryctolagus cuniculus L.)

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

Transgenic Eimeria tenella Expressing Enhanced Yellow Fluorescent Protein Targeted to Different Cellular Compartments Stimulated Dichotomic Immune Responses in Chickens

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