Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation.
Split-CreERT2: temporal control of DNA recombination mediated by split-Cre protein fragment complementation.
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裂解2：通过分裂蛋白片段互补介导的DNA重组的时间控制。
DOI：
10.1371/journal.pone.0008354
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发表时间：
2009-12-16
期刊：
影响因子：
3.7
通讯作者：
Hirrlinger PG
中科院分区：
文献类型：
--
作者：
Hirrlinger J;Requardt RP;Winkler U;Wilhelm F;Schulze C;Hirrlinger PG
DNA重组技术，如Cre/LoxP系统，通过允许体内条件基因调控推进现代生物学研究。然而，在给定时间点精确靶向特定细胞类型仍然具有挑战性，因为空间特异性迄今为止完全取决于驱动Cre重组酶表达的启动子。我们最近建立了分裂Cre，允许DNA重组控制的巧合活动的两个启动子，从而增加空间特异性的Cre介导的DNA重组。为了允许时间控制分裂Cre介导的DNA重组，我们现在通过将分裂Cre蛋白与来自CreERT 2的他莫昔芬诱导型ERT 2结构域融合来扩展分裂Cre。在分裂CreERT 2系统中，Cre介导的DNA重组由两个表达盒以及他莫昔芬应用的时间控制。通过在培养的细胞中使用两种独立的Cre依赖性报告基因，NCre-ERT 2 + ERT 2-CCre的组合被鉴定为具有所有测试的构建体中最有利的性质，显示出约10的诱导比和10 nM至70 nM的4-羟基-他莫昔芬的EC 50值。这些特点的分裂CreERT 2在体外表明，分裂CreERT 2将非常适合于诱导DNA重组在活的小鼠携带LoxP侧翼的等位基因。通过这种方式，split-CreERT 2将提供现代遗传学的新工具，允许对由两个启动子的同时活性定义的细胞群体进行空间和时间精确的遗传访问。
DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2. In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC50-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM. These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.
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影响因子：
4.6
作者：
Hameyer, Dorothe;Loonstra, Ate;Bockamp, Ernesto
通讯作者：
Bockamp, Ernesto
影响因子：
3.5
作者：
Leone, DP;Genoud, S;Suter, U
通讯作者：
Suter, U
DOI：
10.1006/bbrc.1997.7124
发表时间：
1997-08-28
影响因子：
3.1
作者：
Feil, R;Wagner, J;Chambon, P
通讯作者：
Chambon, P
影响因子：
14.9
作者：
Indra, AK;Warot, X;Metzger, D
通讯作者：
Metzger, D
影响因子：
5.3
作者：
Brockschnieder, D;Lappe-Siefke, C;Riethmacher, D
通讯作者：
Riethmacher, D