Gene trapping: an antibody-dependent approach for verifying integration in your favorite gene

Cell Mol Biol Lett. 2008;13(4):614-20. doi: 10.2478/s11658-008-0028-3. Epub 2008 Jul 9.

Abstract

Gene trapping is used to introduce genome-wide insertional mutations in embryonic stem cells. Determining the integration site is based on highthroughput PCR, which has inevitable possibilities for mistakes, thus necessitating clone verification prior to the generation of mutant mice. Here, we propose a rapid method to validate gene identity based on the fact that many high throughput gene-trapping integrations result in fusion proteins encompassing the N-terminal portion of the gene of interest and LacZ being expressed in embryonic stem cells. Our method utilizes an immunoprecipitation assay using a specific N-terminal-directed antibody to the protein product of the gene of interest followed by a color LacZ assay of the immunoprecipitate, strongly supporting the formation of a fusion protein when the color develops.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / metabolism*
  • Cell Line
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / physiology*
  • Genome
  • Humans
  • Mice
  • Mutagenesis, Insertional / methods*
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Antibodies
  • Recombinant Fusion Proteins