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Stable transfection and continuous expression of heterologous genes in Entamoeba invadens
Stable transfection and continuous expression of heterologous genes in Entamoeba invadens
- Source :
- Molecular and biochemical parasitology. 184(1)
- Publication Year :
- 2012
-
Abstract
- Amoebiasis is spread by the ingestion of dormant Entamoeba histolytica cysts. Intervention of encystation could break the transmission cycle, thereby reducing disease burden. The model system used to study trophozoite to cyst differentiation is Entamoeba invadens . Here we describe an electroporation-based method for stable transfection of E. invadens with a plasmid p Ei NEO-LUC containing the neomycin phosphotransferase gene under the control of E. invadens ribosomal protein gene S10 promoter. The plasmid also contains luciferase reporter gene expressed from the promoter of ribosomal protein gene L3. After electroporation, cells receiving the plasmid were selected by growth in 10 μg ml −1 G418 and stable lines were obtained in four to five weeks. The plasmid was replicated episomally to ∼10 copies per haploid genome. In the absence of drug selection 50% of the plasmid copies were lost in 9–10 days. In cells growing under drug selection the reporter gene was continuously expressed throughout the differentiation process from trophozoite to cyst and back, making this system suitable for analysis of genes involved in differentiation.
- Subjects :
- Reporter gene
Electroporation
Genes, Protozoan
Genetic Vectors
Heterologous
Gene Expression
Biology
Transfection
Molecular biology
Entamoeba invadens
Recombinant Proteins
Microbiology
Entamoeba
Plasmid
Ribosomal protein
Genes, Reporter
parasitic diseases
Humans
Parasitology
Luciferase
Luciferases
Molecular Biology
Gene
Plasmids
Subjects
Details
- ISSN :
- 18729428
- Volume :
- 184
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Molecular and biochemical parasitology
- Accession number :
- edsair.doi.dedup.....18cf0540c7642b6ea18085e25ca8eb63