A regulatable transgene expression system for cultured Plasmodium falciparum parasites. Academic Article uri icon

Overview

MeSH

  • Animals
  • Gene Dosage
  • Gene Expression
  • Gene Expression Regulation
  • Genes, Reporter
  • Genetic Complementation Test
  • Luciferases
  • Plasmids
  • Promoter Regions, Genetic

MeSH Major

  • Animals, Genetically Modified
  • Molecular Biology
  • Plasmodium falciparum

abstract

  • The ability to transfect and create transgenic cultured malaria parasites has transformed the study of Plasmodium falciparum over the last decade. With the completion of the annotated genome sequence, the process of gene discovery now routinely includes gene knockouts, over-expression and complementation analysis. However, while this technology has proven extremely valuable, significant limitations exist. In particular, P. falciparum DNA is often unstable and difficult to clone because of its AT-rich, repetitive nature. As a result, transgene expression constructs can be difficult to assemble due to the need to include two expression cassettes on a single plasmid, one to drive expression of the transgene of interest and a second for expression of the selectable marker. In addition, transgene expression levels are usually not regulatable, making it difficult to assess phenotypes that are sensitive to the amount of protein expressed. A plasmid based system for transgene expression is described that uses a single, bidirectional promoter to drive expression of both the transgene and the selectable marker, thus greatly reducing the size of the construct and enhancing stability. Further, by altering the concentration of drug used for selection, it is possible to modulate the copy number of the concatameric episomes and thereby regulate the expression level of the transgene through a range greater than 10 fold. The transgene expression system described here should prove useful for both routine protein over-expression and complementation experiments as well as for experiments in which precisely manipulating the expression level of candidate proteins is desirable. This should provide an additional level of precision to the tools used to study the molecular biology of malaria parasites.

publication date

  • 2008

has subject area

  • Animals
  • Animals, Genetically Modified
  • Gene Dosage
  • Gene Expression
  • Gene Expression Regulation
  • Genes, Reporter
  • Genetic Complementation Test
  • Luciferases
  • Molecular Biology
  • Plasmids
  • Plasmodium falciparum
  • Promoter Regions, Genetic

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2409362

Digital Object Identifier (DOI)

  • 10.1186/1475-2875-7-86

PubMed ID

  • 18492282

Additional Document Info

start page

  • 86

volume

  • 7