Journal article
Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica
International journal for parasitology, Vol.46(3), pp.205-212
03/01/2016
DOI: 10.1016/j.ijpara.2015.11.004
PMID: 26747561
Abstract
Entamoeba histolytica has a robust endogenous RNA interference (RNAi) pathway. There are abundant 27 nucleotide (nt) anti-sense small RNAs (AS sRNAs) that target genes for silencing and the genome encodes many genes involved in the RNAi pathway such as Argonaute proteins. Importantly, an E. histolytica gene with numerous AS sRNAs can function as a "trigger" to induce silencing of a gene that is fused to the trigger. Thus, the amebic RNAi pathway regulates gene expression relevant to amebic biology and has additionally been harnessed as a tool for genetic manipulation. In this study we have further improved the trigger-induced gene silencing method. We demonstrate that rather than using the full-length gene, a short portion of the coding region fused to a trigger is sufficient to induce silencing; the first 537 bp of the E. histolytica rhomboid gene (EhROM1) fused in-frame to the trigger was sufficient to silence EhROM1. We also demonstrated that the trigger method could silence two amebic genes concomitantly; fusion of the coding regions of EhROM1 and transcription factor, EhMyb, in-frame to a trigger gene resulted in both genes being silenced. Alternatively, two genes can be silenced sequentially: EhROM1-silenced parasites with no drug selection plasmid were transfected with trigger-EhMyb, resulting in parasites with both EhROM1 and EhMyb silenced. With all approaches tested, the trigger-mediated silencing was substantive and silencing was maintained despite loss of the G418 selectable marker. All gene silencing was associated with generation of AS 5RNAs to the silenced gene. We tested the reversibility of the trigger system using inhibitors of histone modifications but found that the silencing was highly stable. This work represents a technical advance in the trigger gene silencing method in E. histolytica. Approaches that readily silence multiple genes add significantly to the genetic toolkit available to the ameba research community. (C) 2015 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Details
- Title: Subtitle
- Technical advances in trigger-induced RNA interference gene silencing in the parasite Entamoeba histolytica
- Creators
- Mohamed I. Khalil - National Research CentreBardees M. Foda - National Research CentreSusmitha Suresh - Stanford MedicineUpinder Singh - Stanford University
- Resource Type
- Journal article
- Publication Details
- International journal for parasitology, Vol.46(3), pp.205-212
- DOI
- 10.1016/j.ijpara.2015.11.004
- PMID
- 26747561
- NLM abbreviation
- Int J Parasitol
- ISSN
- 0020-7519
- eISSN
- 1879-0135
- Publisher
- Elsevier
- Number of pages
- 8
- Grant note
- R21AI102277 / NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Allergy & Infectious Diseases (NIAID) AI102277 / National Institutes of Health, USA; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 03/01/2016
- Academic Unit
- Internal Medicine
- Record Identifier
- 9985244553302771
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