Activation kinetics and off-target effects of thymus-initiated cre transgenes.
Activation kinetics and off-target effects of thymus-initiated cre transgenes.
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DOI：
10.1371/journal.pone.0046590
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Petrie HT
中科院分区：
文献类型：
--
作者：
Shi J;Petrie HT
噬菌体酶 Cre 是一种位点特异性重组酶，广泛用于以谱系特异性方式删除 loxP 侧翼 DNA 序列。已经建立了几种将 Cre 表达引导至胸腺中淋巴祖细胞的小鼠品系，但缺乏对它们何时首次变得活跃和/或它们在不同发育阶段的相对效率的并列比较。在这项研究中，我们在四种常见的具有胸腺启动启动子（Lck、Cd2 或 Cd4）的 Cre 转基因菌株中评估了这些。我们发现，虽然它们最终都标记了几乎所有胸腺细胞，但它们的动力学却截然不同，并且除了 Cd4[Cre] 之外，它们并没有忠实地重现相应内源基因的表达模式。也许更重要的是，虽然某些品系的胸腺与对照（Cre 阴性）小鼠的胸腺相比，我们发现 Cre 表达也可能导致脱靶效应，包括胸腺细胞结构中度至重度减少。这些效应发生在缺乏 loxP 侧翼 DNA 靶基因的情况下，并且是剂量和拷贝数依赖性的。细胞结构的丧失可归因于CD4+8+未成熟细胞的特定减少，并且对应于程序性细胞死亡率的增加。除了对胸腺引发的 Cre 转基因的激活动力学进行全面分析外，我们的数据表明，Cre 对 CD4+8+ 细胞具有剂量依赖性毒性，并强调胸腺引发的 Cre 菌株的选择对于最大限度地减少 Cre 的脱靶效应至关重要。
The bacteriophage enzyme Cre is a site-specific recombinase widely used to delete loxP-flanked DNA sequences in lineage-specific fashion. Several mouse lines that direct Cre expression to lymphoid progenitors in the thymus have been established, but a side-by-side comparison of when they first become active, and/or their relative efficiency at various developmental stages, has been lacking. In this study, we evaluated these in four common Cre transgenic strains with thymus-initiated promoters (Lck, Cd2, or Cd4). We found that while all of them eventually labeled nearly all thymocytes, their kinetics were dramatically different, and other than Cd4[Cre], did not faithfully recapitulate the expression pattern of the corresponding endogenous gene. Perhaps even more importantly, while thymuses from some strains compared favorably to thymuses from control (Cre-negative) mice, we found that Cre expression could also result in off-target effects, including moderate to severe decreases in thymic cellularity. These effects occurred in the absence of loxP-flanked DNA target genes, and were dose and copy number dependent. Loss of cellularity was attributable to a specific decrease in CD4+8+ immature cells, and corresponds to an increased rate of programmed cell death. In addition to a comprehensive analysis of activation kinetics in thymus-initiated Cre transgenes, our data show that Cre is toxic to CD4+8+ cells in a dose-dependent fashion, and emphasize that the choice of thymus-initiated Cre strain is critically important for minimizing off-target effects of Cre.
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