Targeted integration of transgenes at the mouse Gt(ROSA)26Sor locus

Abstract

The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase.