Nickerson H D, Colledge W H
Department of Physiology, University of Cambridge, Downing Street, Cambridge, UK.
Gene Ther. 2003 Sep;10(18):1584-91. doi: 10.1038/sj.gt.3302049.
Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.
已证明小于1千碱基（kb）的合成寡核苷酸和DNA片段可在培养的哺乳动物细胞和体内引起位点特异性基因改变。我们使用了一个lacZ报告基因系统来比较人胚肾上皮细胞（HEK293）、中国仓鼠卵巢成纤维细胞（CHOK1）、人支气管上皮细胞（16HBE）和小鼠胚胎干细胞（ES）中游离型和染色体基因修复的效率。lacZ基因包含一个从G到A的核苷酸变化（谷氨酸到赖氨酸突变），该变化消除了β-半乳糖苷酶活性。我们比较了不同基因修复方法纠正此突变并恢复β-半乳糖苷酶活性的效率。我们评估了0.52 - 1.9 kb的PCR生成的双链DNA片段、含有内部硫代磷酸酯连接的20、35或80个碱基的单链DNA寡核苷酸，以及一个68个碱基的RNA:DNA寡核苷酸。所有这些寡核苷酸和DNA片段在游离型质粒中都显示出一定的基因修复能力。0.52 kb或更大的短DNA片段在游离型基因修复中频率最高，而单链DNA寡核苷酸在染色体修复中频率最高。在染色体靶点的情况下，反义DNA寡核苷酸的基因修复频率比其正义对应物高5倍。RNA:DNA嵌合寡核苷酸在染色体或游离型靶点上几乎没有或没有基因修复作用。
