Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis:: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases
Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis:: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases
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DOI：
10.1093/nar/27.22.4324
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发表时间：
1999-11-15
影响因子：
14.9
通讯作者：
Metzger, D
中科院分区：
文献类型：
--
作者：
Indra, AK;Warot, X;Metzger, D
两个LoxP位点之间的条件性DNA切除可以使用Cre-ERT在小鼠中实现，Cre-ERT是人雌激素受体（ER）的突变配体结合结构域与Cre重组酶之间的融合蛋白，Cre重组酶的活性可以由4-羟基-他莫昔芬（OHT）而不是天然ER配体诱导。我们最近表征了新的配体依赖性重组酶Cre-ERT 2，这类似于在培养的细胞中OHT比Cre-ERT更有效地诱导4倍。为了比较这两种配体诱导型重组酶产生暂时控制的体细胞突变的体内效率，我们已经工程化了在表皮基底细胞层中特异性活性的牛角蛋白5启动子的中心下表达LoxP侧翼（floxed）转基因报告基因和Cre-ERT或Cre-ERT 2的转基因小鼠。未检测到背景重组酶活性，而OHT给药后在基底角质形成细胞中诱导重组。有趣的是，一项剂量反应研究显示，Cre-ERT 2对OHT诱导的敏感性是Cre-ERT的10倍。
Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ERT, a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands, We have recently characterized a new ligand-dependent recombinase, Cre-ERT2, which was similar to 4-fold more efficiently induced by OHT than Cre-ERT in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ERT or Cre-ERT2 under the central of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ERT2 was similar to 10-fold more sensitive to OHT induction than Cre-ERT.