van Brabant A J, Williams J K, Parekh-Olmedo H, Kmiec E B
NaPro BioTherapeutics, Genomics Division, Newark, DE, USA.
Pharmacogenomics J. 2004;4(3):175-83. doi: 10.1038/sj.tpj.6500237.
A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.
人类基因中的单核苷酸多态性（SNP）可改变相应蛋白质的行为，进而影响个体对药物治疗的反应。在此，我们描述了一种新颖的双靶向方法，通过使用修饰的单链寡核苷酸（MSSO）将任意选择的SNP引入几乎任何基因中。我们利用该策略在酵母人工染色体（YAC）所含的人类基因中创建SNP。在双靶向方案中，设计两个不同的MSSO以在同一细胞中编辑两个不同的碱基。其中一个基因的变化是选择性的，而另一个是非选择性的。我们表明，通过选择压力鉴定出的群体富含在非选择位点带有编辑碱基的细胞。含有特定SNP或单倍型的人类基因组插入片段的YAC可用于细胞系和转基因动物的药物基因组学应用。
