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Open Access Highly Accessed Methodology article

Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

Alicia S Linford1, Heriberto Moreno1, Katelyn R Good2, Hanbang Zhang3, Upinder Singh34 and William A Petri125*

Author Affiliations

1 Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA

2 Department of Medicine, University of Virginia, Charlottesville, Virginia, USA

3 Department of Internal Medicine, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California, USA

4 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

5 Department of Pathology, University of Virginia, Charlottesville, Virginia, USA

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BMC Microbiology 2009, 9:38  doi:10.1186/1471-2180-9-38

Published: 17 February 2009

Abstract

Background

Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica.

Results

An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.

Conclusion

Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.