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High‐throughput knock‐in coupling gene targeting with the HPRT minigene and Cre‐mediated recombination
Author(s) -
Kim Tae Moon,
Choi Yong Jun,
Ko Jun Ho,
Hasty Paul
Publication year - 2008
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/dvg.20439
Subject(s) - minigene , biology , recombinase , genetics , gene , gene knockin , homologous recombination , gene targeting , cre recombinase , computational biology , transgene , rna , recombination , rna splicing , genetically modified mouse
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.