A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse
A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse
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DOI：
10.1038/nbt811
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发表时间：
2003-05-01
影响因子：
46.9
通讯作者：
Ghyselinck, NB
中科院分区：
文献类型：
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作者：
Schn端tgen, F;Doerflinger, N;Ghyselinck, NB
功能冗余、补偿机制和致死表型通常通过在小鼠中产生生殖系无效突变来阻止对基因功能的全面分析（1）。使用位点特异性重组酶，如催化loxP位点之间重组的Cre（2），可以对携带靶向体细胞突变的小鼠进行工程改造，这些突变既受时间控制，又受细胞类型限制（1，3）。存在许多表达Cre的小鼠品系，但只有少数转基因品系具有其表达依赖于Cre介导事件的报告基因（3）。此外，它们在单个细胞水平上监测基因消融的用途通常是有限的，因为在某些组织中，报告基因可能被沉默（1），受到位置效应杂色的影响（4），或驻留在重组不可及的染色质构型中。因此，不能有效地从报告转基因的表达外推到给定基因的条件等位基因的相同消融模式。通过结合Cre重组酶根据侧翼loxP位点的方向反转或切除DNA片段的能力（6），以及野生型（WT）和突变型loxP位点的可用性（7），我们设计了Cre依赖性遗传开关（FLEx开关），通过该开关，给定基因的表达被关闭，我们证明了这种开关的效率和可靠性，很容易检测，在小鼠中，在单细胞水平上，Cre介导的基因消融。我们将讨论如何使用这种策略以有条件的方式产生遗传修饰。
Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse(1). The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites(2), has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted(1,3). Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event(3). Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced(1), be affected by position-effect variegation(4), or reside in a chromatin configuration inaccessible for recombinations. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites(6), and the availability of both wild-type (WT) and mutant loxP sites(7), we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.