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


CONCLUSIONS
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.
Linford
Alicia S
AS
Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA. asl2c@virginia.edu
Moreno
Heriberto
H
Good
Katelyn R
KR
Zhang
Hanbang
H
Singh
Upinder
U
Petri
William A
WA
D000818:Animals; D004748:Entamoeba histolytica / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017125:Genes, Protozoan; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA; D034622:RNA Interference; D016053:RNA, Protozoan / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
D015800:Protozoan Proteins; C034477:RNA primers; D016053:RNA, Protozoan; D034741:RNA, Small Interfering; D012313:RNA