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Genesis. 2008 Dec;46(12):732-7. doi: 10.1002/dvg.20439.

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

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The Department of Molecular Medicine, The Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.


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 (SAbetageo 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 3beta (Top3beta) 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.

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