International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 76 - GENE ENTRAPMENT STRATEGY FOR PROTEOMICS

A Osipovich
Vanderbilt University

Ruley E
Vanderbilt University

To facilitate genetic and biochemical analysis of mammalian gene functions we have developed a gene entrapment strategy that uses a new gene trap vector LNPAT1. The vector functions as a poly(A) trap targeting most genes regardless whether they are expressed in ES cells. Disrupted genes are identified by sequencing of 3íRACE products which are used for preparation of DNA microarray chips providing a resource to study expression profiles of genes in the mutant library. The LNPAT1 vector incorporates an enhanced green fluorescent protein (EGFP) reporter to monitor gene expression in vitro and in tissues of an intact animal. The vector also contains heterotypic loxP sites for Cre-mediated cassette replacement allowing integrated vectors and genes trapped in ES cells to be engineered for a variety of applications, e.g.: a) to optimise poly(A) gene trap vectors;b) to reverse null mutations;c) to create heteromorphic alleles of disrupted genes;d) for chromosome engineering;e)† to affinity-tag cellular proteins to characteriseinteracting proteins from normal tissues of the mouse.Depending on what part of gene is disrupted with LNPAT1 vector replacement with N-TAP or C-TAP affinity tag vectors with splice sites in corresponding reading frames allows to create a genome wide resource for study of protein-protein interactions.


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