Back to Search
Start Over
piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella.
- Source :
-
PloS one [PLoS One] 2012; Vol. 7 (6), pp. e40075. Date of Electronic Publication: 2012 Jun 29. - Publication Year :
- 2012
-
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.
- Subjects :
- Animals
Animals, Genetically Modified
Bacterial Proteins metabolism
Base Sequence
Eimeria tenella cytology
Flow Cytometry
Genome, Protozoan genetics
Luminescent Proteins metabolism
Molecular Sequence Data
Mutagenesis, Insertional genetics
Oocysts cytology
Oocysts metabolism
Parasites cytology
Plasmids genetics
Reproducibility of Results
DNA Transposable Elements genetics
Eimeria tenella genetics
Gene Transfer Techniques
Parasites genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 7
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 22768223
- Full Text :
- https://doi.org/10.1371/journal.pone.0040075