Spontaneous recombinase activity of Cre-ERT2 in vivo.

Spontaneous recombinase activity of Cre-ERT2 in vivo.
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DOI:
10.1007/s11248-017-0018-1
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发表时间:
2017-06
影响因子:
3
通讯作者:
Blank, Robert D.
Blank, Robert D.
中科院分区:
生物学4区
文献类型:
--
作者:
Kristianto, Jasmin;Johnson, Michael G.;Zastrow, Ryley K.;Radcliff, Abigail B.;Blank, Robert D.

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诱导型Cre-ERT重组酶技术广泛用于基因打靶研究。第二代诱导型Cre-ERT重组酶,半合子B6. 129 S-Tg(UBC-cre/ERT 2)1 Ejb/J(以下简称为Cre-ERT 2),突变的雌激素受体和Cre重组酶的融合物,被工程改造成比原始Cre-ERT更有效和特异性。选择性Cre介导的重组的假定机制是Cre在基础状态下在细胞质中的隔离,仅在他莫昔芬存在下易位到细胞核。我们利用报告小鼠(B6.129(Cg)-Gt(ROSA)26 Sortm 4(ACTB-tdTomato,-EGFP)Luo/J)和内皮素转换酶-1(Ece 1)floxed转基因小鼠系来评估Cre-ERT 2活性。我们观察到自发Cre活动在这两种设置。非预期Cre活动是一个混杂因素,对数据解释有潜在的巨大影响。因此,在实验设计中考虑背景Cre活性是重要的。
Inducible Cre-ERT recombinase technology is widely used for gene targeting studies. The second generation of inducible Cre-ERT recombinase, hemizygous B6.129S-Tg(UBC-cre/ERT2)1Ejb/J (hereafter abbreviated as Cre-ERT2), a fusion of a mutated estrogen receptor and Cre recombinase, was engineered to be more efficient and specific than the original Cre-ERT. The putative mechanism of selective Cre-mediated recombination is Cre sequestration in the cytoplasm in the basal state with translocation to the nucleus only in the presence of tamoxifen. We utilized both a reporter mouse (B6.129 (Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) and endothelin converting enzyme-1 (Ece1) floxed transgenic mouse line to evaluate Cre-ERT2 activity. We observed spontaneous Cre activity in both settings. Unintended Cre activity is a confounding factor that has a potentially large impact on data interpretation. Thus, it is important to consider background Cre activity in experimental design.
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