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

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.

Abbreviations

β-geo:

fusion of β-galactosidase and neomycin transferase

DCLK2:

doublecortin-like-kinase 2

DCX:

doublcortin

DsRed:

Discosoma sp. reef coral red fluorescent protein

ES:

embryonic stem

HEK293:

human embryonic kidney cell line

IGTC:

International Gene Trap Consortium

IP:

immunoprecipitation

LacZ:

beta galactosidase reporter gene

PCR:

polymerase chain reaction

5′-RACE:

Rapid amplification of 5′ complementary DNA ends

X-gal:

5-bromo-4-chloro-3-indolyl-β-Dgalactoside

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Correspondence to Orly Reiner.

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Gorelik, A., Sapir, T. & Reiner, O. Gene trapping: An antibody-dependent approach for verifying integration in your favorite gene. Cell Mol Biol Lett 13, 614–620 (2008). https://doi.org/10.2478/s11658-008-0028-3

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  • DOI: https://doi.org/10.2478/s11658-008-0028-3

Key words

  • Gene-trap
  • LacZ fusion protein
  • Integration site