High‐throughput knock‐in coupling gene targeting with the HPRT minigene and Cre‐mediated recombination

Single nucleotide polymorphisms (SNPs) may influence protein function possibly contributing to phenotype; yet, for most SNPs their potential influence is unknown. Here, we present a technique in mouse embryonic stem cells that enables high‐throughput knock‐in (the placement of coding sequences adjacent to a specific endogenous promoter). Our methodology utilizes gene targeting with a combination of two selection cassettes (SAβgeo and the HPRT minigene) along with site‐specific recombinases (Cre/loxP and FLP/FRT) to efficiently introduce multiple DNA sequences, including enhanced green fluorescent protein (eGFP), adjacent to the DNA topoisomerase 3β ( Top3β ) promoter. This technology enables rapid and efficient introduction of DNA sequences to a specific location and advances high‐throughput analysis of many SNPs with control for expression and genetic background. genesis 46:732–737, 2008. © 2008 Wiley‐Liss, Inc.