CONDITIONAL SITE-SPECIFIC RECOMBINATION IN MAMMALIAN-CELLS USING A LIGAND-DEPENDENT CHIMERIC CRE RECOMBINASE
CONDITIONAL SITE-SPECIFIC RECOMBINATION IN MAMMALIAN-CELLS USING A LIGAND-DEPENDENT CHIMERIC CRE RECOMBINASE
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DOI：
10.1073/pnas.92.15.6991
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发表时间：
1995-07-18
影响因子：
11.1
通讯作者：
CHAMBON, P
中科院分区：
文献类型：
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作者：
METZGER, D;CLIFFORD, J;CHAMBON, P
我们开发了一种策略，通过使用融合蛋白Cre-ER，以有条件的方式在哺乳动物细胞中产生突变基因，该融合蛋白Cre-ER由与人雌激素受体的配体结合域连接的loxP位点特异性Cre重组酶组成。我们已经建立了组成性表达Cre-ER的纯合子类维生素A X受体α阴性（RXR α（-/-））F9胚胎癌细胞，并且已经表明雌二醇或雌激素激动剂/拮抗剂4羟基他莫昔芬有效地诱导重组酶活性，而在不存在配体或存在抗雌激素ICI 164，384的情况下没有检测到活性。此外，使用含有侧翼为loxP位点的选择标记的靶向载体，我们已经使这样的系中的一个视黄酸受体α等位基因失活，证明重组酶的存在不抑制同源重组。将这种条件性位点特异性重组系统与Cre-ER的组织特异性表达相结合可以允许以时空调节的方式在体内修饰哺乳动物基因组。
We have developed a strategy to generate mutant genes in mammalian cells in a conditional manner by employing a fusion protein, Cre-ER, consisting of the loxP site-specific Cre recombinase linked to the ligand-binding domain of the human estrogen receptor. We have established homozygous retinoid X receptor alpha-negative (RXR alpha(-/-)) F9 embryonal carcinoma cells constitutively expressing Cre-ER and have shown that estradiol or the estrogen agonist/antagonist 4 hydroxytamoxifen efficiently induced the recombinase activity, whereas no activity was detected in the absence of ligand or in the presence of the antiestrogen ICI 164,384. Furthermore, using a targeting vector containing a selection marker flanked by loxP sites, we have inactivated one retinoic acid receptor alpha allele in such a line, demonstrating that the presence of the recombinase does not inhibit homologous recombination. Combining this conditional site-specific recombination system with tissue-specific expression of Cre-ER may allow modification of the mammalian genome in vivo in a spatiotemporally regulated manner.