Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.
Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.
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DOI：
10.1186/1472-6750-13-87
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发表时间：
2013-10-20
影响因子：
3.5
通讯作者：
Brown W
中科院分区：
文献类型：
--
作者：
Xu Z;Thomas L;Davies B;Chalmers R;Smith M;Brown W
噬菌体编码的丝氨酸整合酶，如φC31整合酶，广泛用于基因组工程。已经描述了15种这样的整合酶，但它们用于基因组工程的效用尚未在统一的测定中进行比较。我们比较了15种丝氨酸整合酶在哺乳动物细胞中的DNA操作效用，首先证明了它们在大肠杆菌中的功能。杆菌染色体重组报告显示，7个整合酶在人成纤维细胞和小鼠胚胎干细胞的染色体整合DNA上有活性。其余八种酶中的五种对染色体外底物有活性，从而证明介导染色体外重组的能力并不指导介导整合DNA上的位点特异性重组的能力。对整合的DNA有活性的所有整合酶也促进不通过保守位点特异性重组介导的DNA整合反应或破坏重组位点，但这些异常反应的程度至少在一个数量级上变化。Bxb1整合酶产生的重组体大约是第二种最好的重组酶φ C31整合酶的两倍，对重组位点的损伤也大约是第二种最好的重组酶φC31整合酶的两倍。我们的结论是Bxb1和φC31整合酶是脊椎动物细胞基因组工程的首选试剂，DNA损伤修复是这类位点特异性重组酶应用的主要限制。
Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase.
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DOI：
10.1073/pnas.95.10.5505
发表时间：
1998-05-12
影响因子：
11.1
作者：
Thorpe, HM;Smith, MCM
通讯作者：
Smith, MCM
影响因子：
3.7
作者：
Yamaguchi S;Kazuki Y;Nakayama Y;Nanba E;Oshimura M;Ohbayashi T
通讯作者：
Ohbayashi T
影响因子：
2.9
作者：
Seibler, J;Bode, J
通讯作者：
Bode, J
通过将PHIC31积分酶和CRE重组酶的活性结合的大型和复杂转基因的体内组装。
影响因子：
14.9
作者：
Dafhnis-Calas, F;Xu, ZY;Haines, S;Malla, SK;Smith, MCM;Brown, WRA
通讯作者：
Brown, WRA
影响因子：
3.7
作者：
Canchaya, C;Desiere, F;Brüssow, H
通讯作者：
Brüssow, H