Yáñez R J, Porter A C
Gene Targeting Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, DuCane Road, London W12 0NN, UK.
Gene Ther. 1999 Jul;6(7):1282-90. doi: 10.1038/sj.gt.3300945.
The ideal therapy for single gene disorders would be repair of the mutated disease genes. Homologous recombination is one of several cellular mechanisms for the repair of DNA damage. Recombination between exogenous DNA and homologous chromosomal loci (gene targeting) can be used to repair an endogenous gene, but the low efficiency of this process is a serious barrier to its therapeutic potential. Recent progress in the isolation and characterisation of mammalian genes and proteins involved in DNA recombination has raised the possibility that the cellular biochemistry of recombination can be manipulated to improve the efficiency of gene targeting. As an initial test of this approach, we have overexpressed the gene encoding hRAD51, a protein with homologous DNA pairing and strand exchange activities, in human cells and measured its effect on gene targeting. We report a two- to three-fold increase in gene targeting, and enhanced resistance to ionising radiation in hRAD51-overexpressing cells with no obvious detrimental effects. These observations provide valuable genetic evidence for the involvement of hRAD51 in both gene targeting and DNA repair in human cells. Our data also establish overexpression of recombination genes as a viable approach to improving gene targeting efficiencies.
单基因疾病的理想治疗方法是修复突变的致病基因。同源重组是DNA损伤修复的几种细胞机制之一。外源DNA与同源染色体位点之间的重组（基因打靶）可用于修复内源基因，但该过程的低效率严重阻碍了其治疗潜力。在参与DNA重组的哺乳动物基因和蛋白质的分离与表征方面的最新进展，提高了通过操纵重组的细胞生物化学来提高基因打靶效率的可能性。作为该方法的初步测试，我们在人类细胞中过表达了编码hRAD51的基因，hRAD51是一种具有同源DNA配对和链交换活性的蛋白质，并测量了其对基因打靶的影响。我们报告说，在过表达hRAD51的细胞中，基因打靶增加了两到三倍，并且对电离辐射的抗性增强，且没有明显的有害影响。这些观察结果为hRAD51参与人类细胞的基因打靶和DNA修复提供了有价值的遗传学证据。我们的数据还确立了重组基因的过表达是提高基因打靶效率的一种可行方法。
