Olsen P A, McKeen C, Krauss S
Section for Genetic Therapy, Institute of Microbiology, The National Hospital, Oslo, Norway.
Gene Ther. 2003 Oct;10(21):1830-40. doi: 10.1038/sj.gt.3302079.
Branched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3-6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of b-oligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.
基于一种新型分支单体的分支寡核苷酸（b-寡核苷酸）被用于体内位点特异性序列改变。在中国仓鼠卵巢细胞中，利用稳定整合的突变型增强绿色荧光蛋白（EGFP）模板，用b-寡核苷酸转染后，计数发现高达0.1%的细胞为EGFP阳性。通过抗EGFP免疫细胞化学证明了转化细胞中存在EGFP蛋白。对转化细胞进行基因组测序，在40%的分析克隆中显示校正后的野生型密码子，而9.3%的序列显示校正后的野生型序列和一个额外的附带突变。尽管基因组位点得到了稳定校正，但转化细胞在3 - 6天后进入选择性凋亡。在同源重组途径缺陷的Irs-1细胞系中，b-寡核苷酸诱导的位点特异性序列转化频率降低。突变细胞系中转化率的降低可通过用XRCC2 cDNA互补得到部分挽救。
