Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus.

abstract Article history:Received 1 May 2008Accepted 17 September 2008Available online 3 December 2008Keywords:Embryonic stem cellsGene targetingVectorsGenomicsHprt1Knock-inMicePromoter regionsTransgenic We have engineered a set of useful tools that facilitate targeted single copy knock-in (KI) at the hypoxanthineguanine phosphoribosyl transferase 1 (Hprt1) locus. We employed fine scale mapping to delineate the precisebreakpoint location at the Hprt1 b-m3 locus allowing allele specific PCR assays to be established. Our suite oftools contains four targeting expression vectors and a complementing series of embryonic stem cell lines.Two of these vectors encode enhanced greenfluorescent protein (EGFP) driven by the humancytomegalovirus immediate-early enhancer/modified chicken beta-actin (CAG) promoter, whereas the othertwo permit flexible combinations of a chosen promoter combined with a reporter and/or gene of choice. Wehave validated our tools as part of the Pleiades Promoter Project (http://www.pleiades.org), with thegeneration of brain-specific EGFP positive germline mouse strains.© 2008 Elsevier Inc. All rights reserved.