Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11

Extremophiles. 2017 Jan;21(1):65-72. doi: 10.1007/s00792-016-0880-4. Epub 2016 Oct 18.

Abstract

Larvae of the African midge Polypedilum vanderplanki show extreme desiccation tolerance, known as anhydrobiosis. Recently, the cultured cell line Pv11 was derived from this species; Pv11 cells can be preserved in the dry state for over 6 months and retain their proliferation potential. Here, we attempted to expand the use of Pv11 cells as a model to investigate the mechanisms underlying anhydrobiosis in P. vanderplanki. A newly developed vector comprising a constitutive promoter for the PvGapdh gene allowed the expression of exogenous proteins in Pv11 cells. Using this vector, a stable Pv11 cell line expressing green fluorescence protein (GFP) was established and retained desiccation tolerance. Gene silencing with GFP-specific siRNAs significantly suppressed GFP expression to approximately 7.5-34.6% of that in the non-siRNA-transfected GFP stable line. Establishment of these functional assays will enable Pv11 cells to be utilized as an effective tool to investigate the molecular mechanisms underlying anhydrobiosis.

Keywords: Anhydrobiosis; Desiccation tolerant cells; Gene expression system; RNAi.

MeSH terms

  • Animals
  • Cell Culture Techniques / methods
  • Cell Line
  • Chironomidae / cytology
  • Chironomidae / genetics*
  • Desiccation*
  • Gene Transfer Techniques*
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / genetics
  • Green Fluorescent Proteins / genetics
  • Insect Proteins / genetics
  • Larva / cytology
  • RNA Interference*
  • Stress, Physiological / genetics

Substances

  • Insect Proteins
  • Green Fluorescent Proteins
  • Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)