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Targeted modification of mammalian genomes

机译：哺乳动物基因组的靶向修饰

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The stable and site-specific modification of mammalian genomes has a variety of applications in biomedicine and biotechnology. Here we outline two alternative approaches that can be employed to achieve this goal: homologous recombination (HR) or site-specific recombination. Homologous recombination relies on sequence similarity (or rather identity) of a piece of DNA that is introduced into a host cell and the host genome. In most cell types, the frequency of homologous recombination is markedly lower than the frequency of random integration. Especially in somatic cells, homologous recombination is an extremely rare event. However, recent strategies involving the introduction of DNA double-strand breaks, triplex forming oligonucleotides or adeno-associated virus can increase the frequency of homologous recombination. Site-specific recombination makes use of enzymes (recombinases, transposases, integrases), which catalyse DNA strand exchange between DNA molecules that have only limited sequence homology. The recognition sites of site-specific recombinases (e.g. Cre, Flp or PhiC31 integrase) are usually 30-50 bp. In contrast, retroviral integrases only require a specific dinucleotide sequence to insert the viral cDNA into the host genome. Depending on the individual enzyme, there are either innumerable or very few potential target sites for a particular integrase/recombinase in a mammalian genome. A number of strategies have been utilised successfully to alter the site-specificity of recombinases. Therefore, site-specific recombinases provide an attractive tool for the targeted modification of mammalian genomes.
机译：哺乳动物基因组的稳定和位点特异性修饰在生物医学和生物技术中具有多种应用。在这里，我们概述了可用于实现此目标的两种替代方法：同源重组（HR）或位点特异性重组。同源重组依赖于引入宿主细胞和宿主基因组中的DNA片段的序列相似性（或同一性）。在大多数细胞类型中，同源重组的频率明显低于随机整合的频率。特别是在体细胞中，同源重组是极为罕见的事件。然而，涉及引入DNA双链断裂，形成三链体的寡核苷酸或腺相关病毒的最新策略可以增加同源重组的频率。位点特异性重组利用酶（重组酶，转座酶，整合酶），该酶催化仅具有有限序列同源性的DNA分子之间的DNA链交换。位点特异性重组酶（例如Cre，Flp或PhiC31整合酶）的识别位点通常为30-50 bp。相反，逆转录病毒整合只需要特定的二核苷酸序列即可将病毒cDNA插入宿主基因组。根据单个酶的不同，哺乳动物基因组中特定整合酶/重组酶的潜在靶位点数众多或很少。已成功利用许多策略来改变重组酶的位点特异性。因此，位点特异性重组酶为哺乳动物基因组的靶向修饰提供了一种有吸引力的工具。

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