Spontaneous excision and facilitated recovery as a control for phenotypes arising from RNA interference and other dominant transgenes

Mol Biochem Parasitol. 2018 Mar:220:42-45. doi: 10.1016/j.molbiopara.2018.01.004. Epub 2018 Feb 3.

Abstract

An essential control for genetic manipulation of microbes is the regeneration of the wild-type state and phenotype to validate that any mutant phenotypes are 'on target'. For Leishmania gene knockouts, this is often done by re-expression of the target gene from episomal vectors, often bearing counter-selectable markers. Methods for similarly validating the outcomes from dominant mutations such as those arising from RNA interference (RNAi) are needed. We present here such an approach, relying on facilitated recovery after spontaneous excision - or 'popouts' - of dominant transgenes stably inserted into the ribosomal RNA array, utilizing GFP as a marker and single cell sorting to recover regenerated WT controls. We validate its utility using RNA interference knockdowns of the paraflagellar rod gene PFR2 of L. (Viannia) braziliensis. The method yields stably modified lines suitable for long term studies of Leishmania virulence, relies solely on host rather than introduced genetic machinery, and is thus readily applied in many species and circumstances including functional genetic testing.

Keywords: Dominant mutant transgenes; Leishmania; Paraflagellar rod; RNA interference; Spontaneous excision; Transfection controls.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Flow Cytometry
  • Gene Deletion*
  • Genes, Reporter
  • Genes, rRNA
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Leishmania / genetics*
  • Leishmania / growth & development
  • Leishmania / isolation & purification
  • Molecular Biology / methods*
  • Mutagenesis, Insertional*
  • Phenotype*
  • RNA Interference*
  • Recombination, Genetic
  • Staining and Labeling
  • Transgenes*

Substances

  • Green Fluorescent Proteins