Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

Alessio Cardinale, Ilaria Filesi, Sonia Mattei, Silvia Biocca

Research output: Contribution to journalArticle

Abstract

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype.

Original languageEnglish
Pages (from-to)171-178
Number of pages8
JournalMethods
Volume34
Issue number2
DOIs
Publication statusPublished - Oct 2004

Keywords

  • Aggresome
  • Fluorescence microscopy
  • Green fluorescent protein
  • Intracellular antibody
  • Proteasome
  • scFv fragment
  • Targeting signal

ASJC Scopus subject areas

  • Molecular Biology

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