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Transgenic Research

, Volume 21, Issue 3, pp 511–521 | Cite as

Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells

  • Raphaël Duvoisin
  • Mary A. Ayuk
  • Gabriel Rinaldi
  • Sutas Suttiprapa
  • Victoria H. Mann
  • Clarence M. Lee
  • Nicola Harris
  • Paul J. Brindley
Original Paper

Abstract

Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral [Murine leukemia virus (MLV)] and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs—stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.

Keywords

Schistosome Schistosoma mansoni Short hairpin RNA U6 gene Promoter Firefly luciferase RNA interference Murine leukemia virus piggyBac transposon 

Notes

Acknowledgments

Schistosome-infected snails were supplied by Dr. Fred Lewis, Biomedical Research Institute, Rockville, MD under National Institutes of Health (NIH), National Institute of Allergy and Infectious Disease (NIAID) contract HHSN272201000005I. These studies were supported by NIH-NIAID award R01AI072773 (the content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or the NIH).

Supplementary material

11248_2011_9548_MOESM1_ESM.ppt (144 kb)
Human U6 gene promoter driving short hairpin RNA (shRNA) in human HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni after transduction with MLV retroviral virions. Panel A: Knockdown of luciferase (49% knockdown) in HT1080 cells transduced with pLNHX_HsU6_shLuc (HsU6 shLuc) virions compared to control (HsU6 Scram) virions. Panel B: left panel: Knockdown (60%) of luciferase in schistosomules transduced with pLNHX_HsU6_shLuc (HsU6 shLuc) virions compared to control (HsU6 Scram) virions, right panel: Knockdown (85%) of luciferase in schistosomules transduced with HsU6 shLuc virions compared to control (HsU6 Scram) virions. Luciferase activity is expressed as relative light units per second per microgram of soluble protein (RLU/s/μg) (PPT 144 kb)
11248_2011_9548_MOESM2_ESM.ppt (145 kb)
Comparison between human U6 and schistosome U6 promoter driving short hairpin RNAs targeting firefly luciferase in a HT1080 cell line stably expressing firefly luciferase and in schistosomules of Schistosoma mansoni after transfection with pXL-Bac II plasmids. Panel A: Knockdown was seen in cells transfected with both the human U6 (81% knockdown) and schistosome U6 (64%) promoters in comparison with mock-treatment control. Less knockdown were apparent in controls transfected with the human U6 and schistosome scrambled target sequence based plasmids (60 and 55% respectively). Panel B: In schistosomules, stronger knockdown was seen with human U6 promoter (62% knockdown, i.e. the average knockdown of duplicate experiments shown in the figure) compared with schistosome U6 promoter (≤0.1% knockdown; i.e. the average knockdown of duplicate experiments shown in the figure) driving shRNA after transfection with pXL-Bac II plasmid constructs. In both panels A and B, luciferase activity is expressed as relative light units per second per microgram of soluble protein (RLU/s/μg) (PPT 145 kb)
11248_2011_9548_MOESM3_ESM.docx (33 kb)
Supplementary material 3 (DOCX 32 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Raphaël Duvoisin
    • 1
    • 2
  • Mary A. Ayuk
    • 1
    • 3
  • Gabriel Rinaldi
    • 1
    • 4
  • Sutas Suttiprapa
    • 1
  • Victoria H. Mann
    • 1
  • Clarence M. Lee
    • 3
  • Nicola Harris
    • 2
  • Paul J. Brindley
    • 1
  1. 1.Department of Microbiology, Immunology & Tropical MedicineThe George Washington University Medical CenterWashingtonUSA
  2. 2.Swiss Vaccine Research Institute and Global Health Institute, Ecole Polytechnique Fédérale de LausanneLausanneSwitzerland
  3. 3.Department of BiologyHoward UniversityWashingtonUSA
  4. 4.Departamento de Genética, Facultad de MedicinaUniversidad de la República, (UDELAR)MontevideoUruguay

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