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Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers

Nature Biotechnology volume 19, pages 231–234 (2001)Cite this article

Abstract

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

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Figure 1: Distribution of antisense biotinylated PNAs in trophozoites of strain HK9 after 2 h (A,B) and 24 h (C,D) incubation with 20 μM PNA.
Figure 2: Neomycin phosphorotransferase activity of lysates of HK9 trophozoites treated with 20 μM NEO17ATG antisense PNA for 96 h.
Figure 3: Erd2 protein levels in lysates of HK9 trophozoites treated with 10 μM ERD17ATG antisense PNA for 96 h.
Figure 4: Effect of PNA treatment on trophozoite growth.

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Acknowledgements

This work was supported in part by CONACyT (Consejo Nacional de Ciencia y Technología) grant 27826-N (Mexico), Dirección General de Asusntos del Personal Académico grant 207097 (Mexico), a collaboration travel grant from the Centro Nazionale per la Ricerca (Italy) and CONACyT (Mexico). We wish to thank Angeles Gutiérrez, Xóchitl Alvarado, Enrico Millo, Gianluca Damonte, and Drs. Paul Lizardi, Mario Zurita and Eduardo Horjales.

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Authors and Affiliations

  1. Instituto de Biotecnología/UNAM. Av. Universidad 2001, Morelos, 62210, Mexico

    Roberto P. Stock, Alejandro Olvera, Ricardo Sánchez, Andrés Saralegui, Rosana Sanchez-Lopez, Marco A. Ramos & Alejandro Alagón

  2. Institute of Biochemistry, University of Genoa, Viale Benedetto XV, I-16132, Italy

    Sonia Scarfì & Umberto Benatti

  3. National Cancer Institute, IST, Largo R. Benzi, Genoa, 16132, Italy

    Lidia C. Boffa

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  1. Roberto P. Stock
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Correspondence to Alejandro Alagón.

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Stock, R., Olvera, A., Sánchez, R. et al. Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers. Nat Biotechnol 19, 231–234 (2001). https://doi.org/10.1038/85671

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