Murphy B R, Moayedpardazi H S, Gewirtz A M, Diamond S L, Pierce E A
Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA.
Gene Ther. 2007 Feb;14(4):304-15. doi: 10.1038/sj.gt.3302866. Epub 2006 Oct 5.
Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10(-4), leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.
单链寡脱氧核苷酸（ssODN）基因靶向技术可能有助于动物模型的创建和基因修复治疗。ssODN的脂质转染可将点突变引入靶基因。然而，在小鼠胚胎干细胞（ESC）中的典型效率<10^(-4)，导致校正过于罕见而无法有效识别。我们开发了整合有突变新霉素抗性基因（Tyr22Ter）的ESC系。用ssODN进行靶向作用后，修复的细胞在G418选择中存活下来。校正效率因不同的脂质转染程序、克隆系和ssODN设计而异，范围为每接种百万个细胞有1至100次校正。使用Cy5标记的ssODN进行细胞分选的摄取研究表明，40%的校正集中在转染效果最佳的22%的细胞中。测试了四种不同的碱基对错配，结果表明错配的碱基特异性至关重要。双错配ssODN也表现出错配偏好。这些ESC系可能有助于开发用于动物生产或离体基因治疗的改进型ssODN靶向技术。
