title: piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella.
abstract: 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.
authors: National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors: Huali
authors: H
authors: Su
authors: Xianyong
authors: X
authors: Liu
authors: Wenchao
authors: W
authors: Yan
authors: Tuanyuan
authors: T
authors: Shi
authors: Xinxin
authors: X
authors: Zhao
authors: Damer P
authors: DP
authors: Blake
authors: Fiona M
authors: FM
authors: Tomley
authors: Xun
authors: X
authors: Suo
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D016786:Eimeria tenella / Q000166:cytology / Q000235:genetics*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D018503:Genome, Protozoan / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D033661:Oocysts / Q000166:cytology / Q000378:metabolism; D010271:Parasites / Q000166:cytology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria