Endogenous gene tagging with fluorescent proteins

Methods Mol Biol. 2015;1239:231-40. doi: 10.1007/978-1-4939-1862-1_12.

Abstract

Human genome manipulation has become a powerful tool for understanding the mechanisms of numerous diseases including cancer. Inserting reporter sequences in the desired locations in the genome of a cell can allow monitoring of endogenous activities of disease related genes. Native gene expression and regulation is preserved in these knock-in cells in contrast to cell lines with target overexpression under an exogenous promoter as in the case of transient transfection or stable cell lines with random integration. The fusion proteins created using the modern genome editing tools are expressed at their physiological level and thus are more likely to retain the characteristic expression profile of the endogenous proteins in the cell. Unlike biochemical assays or immunostaining, using a tagged protein under endogenous regulation avoids fixation artifacts and allows detection of the target's activity in live cells. Multiple gene targets could be tagged in a single cell line allowing for the creation of effective cell-based assays for compound screening to discover novel drugs.

MeSH terms

  • Cell Culture Techniques
  • Cloning, Molecular / methods
  • Gene Expression
  • Gene Fusion*
  • Genes, Reporter*
  • Genetic Engineering / methods*
  • Genetic Vectors / genetics
  • Humans
  • Luminescent Proteins / genetics*
  • Mutagenesis, Insertional*
  • Transfection

Substances

  • Luminescent Proteins