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·         High-throughput method of generating multiple types of alleles of one gene in a site-specific manner

·         Can use to modify up to ~4,500 genes in the mouse genome for which there is an existing compatible gene trap collection

·         Will enable a detailed analysis of gene function through the analysis of multiple kinds of designed alleles

·         Manipulation of a single genomic locus eliminates position effects

·         The recombination process generates an internal control cell line

·         Could use to replace mouse genes with human homologs for drug studies

·         High-throughput method of generating multiple types of alleles of one gene in a site-specific manner

·         Can use to modify up to ~4,500 genes in the mouse genome for which there is an existing compatible gene trap collection

·         Will enable a detailed analysis of gene function through the analysis of multiple kinds of designed alleles

·         Manipulation of a single genomic locus eliminates position effects

·         The recombination process generates an internal control cell line

·         Could use to replace mouse genes with human homologs for drug studies

Background

Since the human genome has been sequenced, researchers are now focusing on defining the roles of each the ~30,000 genes in development and disease. As most human genes have homologs in the mouse genome, many researchers study the function of mouse genes to model the function of related human genes.

To study all aspects of gene function, one must be able to manipulate a single gene to generate many kinds of mutations: deletions, point mutations, insertion of exogenous DNA, etc. In mice, such site-directed mutagenesis is commonly achieved by homologous recombination of embryonic stem cells, which can be laborious, time-consuming, and inefficient. Thus, there is a need for technologies that allow for high-throughput, site-directed mutagenesis.

Description

UCSF investigators have designed vectors and methods for high-throughput genetic modification of an existing collection of gene trap insertions in mouse embryonic stem cells. The collection is publically available from the Sanger Gene Trap Consortium and currently consists of ~20,000 random insertions in ~4,500 genes in the mouse genome.

The vectors designed by UCSF investigators make use of recombinase activity to drive three recombination steps at the site of the gene trap insertion. The first step allows for reversion of the original gene trap insertion, resulting in the generation of an internal control. The final two steps allow for the insertion of new DNA of interest, resulting in the desired mutant or allele. Each recombination step can be selected easily using a visible genetic marker.

This system allows one to generate any number of mutations repeatedly into the same genomic site, with efficiencies as high as 80%. Additionally, because the insertions are controlled by the same endogenous promoter elements, position effects, which can create artifacts by misexpression or overexpression, are effectively eliminated. Using these vectors and methods, one could generate with relative ease a variety of site-directed mutations in any of the 4,500 genes for which there is an available enhancer trap insertion.