Stable transfection and continuous expression of heterologous genes in Entamoeba invadens

Singh, Nishant ; Ojha, Sandeep ; Bhattacharya, Alok ; Bhattacharya, Sudha (2012) Stable transfection and continuous expression of heterologous genes in Entamoeba invadens Molecular and Biochemical Parasitology, 184 (1). pp. 9-12. ISSN 0166-6851

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Official URL: http://www.sciencedirect.com/science/article/pii/S...

Related URL: http://dx.doi.org/10.1016/j.molbiopara.2012.02.012

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 pEiNEO-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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Entamoeba Invadens; Stable Transfection; Encystation; Neomycin Phosphotransferase Gene; Luciferase; Electroporation
ID Code:98041
Deposited On:29 Jan 2014 11:35
Last Modified:29 Jan 2014 11:35

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