Kim Tae Moon, Choi Yong Jun, Ko Jun Ho, Hasty Paul
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.
Genesis. 2008 Dec;46(12):732-7. doi: 10.1002/dvg.20439.
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.
单核苷酸多态性（SNP）可能会影响蛋白质功能，进而可能影响表型；然而，对于大多数SNP而言，其潜在影响尚不清楚。在此，我们介绍一种在小鼠胚胎干细胞中的技术，该技术能够实现高通量敲入（将编码序列置于特定内源性启动子附近）。我们的方法利用基因打靶技术，结合两个选择盒（SAbetageo和HPRT小基因）以及位点特异性重组酶（Cre/loxP和FLP/FRT），有效地将多个DNA序列，包括增强型绿色荧光蛋白（eGFP），引入到DNA拓扑异构酶3β（Top3β）启动子附近。这项技术能够快速、高效地将DNA序列引入特定位置，并推进对许多SNP的高通量分析，同时可控制表达和遗传背景。
