Gene trapping: an antibody-dependent approach for verifying integration in your favorite gene.
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.
Gorelik
Anna
A
Department of Molecular Genetics, The Weizmann Institute of Science, 76100, Rehovot, Israel.
Sapir
Tamar
T
Reiner
Orly
O
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D016678:Genome; D006801:Humans; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D011993:Recombinant Fusion Proteins