Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter
Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter
复制标题
DOI：
10.3389/fimmu.2019.02228
复制
发表时间：
2019-09-20
影响因子：
7.3
通讯作者：
Costalonga, Massimo
中科院分区：
文献类型：
--
作者：
Bittner-Eddy, Peter D.;Fischer, Lori A.;Costalonga, Massimo
将特定的loxp -侧翼基因序列与细胞调控启动子表达的Cre重组酶结合在一起的小鼠模型已经成为研究基因功能的重要工具。然而，关键的是，由于驱动启动子的混杂活性，Cre重组酶的表达可能并不总是局限于感兴趣的靶细胞或组织。Cre重组酶的表达和loxp基因的切除可能发生在非靶细胞中，并且可能不容易明显。在这里，我们报告了通过将il17a或Foxp3启动子与组成性表达的絮凝停止tdTomato报告基因结合，表达的Cre重组酶的保真度。F-1小鼠foxp3驱动的Cre重组酶在Treg细胞和一系列其他免疫细胞中诱导tdTomato红色荧光蛋白。tdTomato的表达频率在淋巴细胞（B细胞和CD8 T细胞）和血液常驻骨髓细胞（树突状细胞、单核细胞、中性粒细胞）之间是可变的，但正相关（p < 0.0001），这表明Foxp3启动子的随机活性，而不是共同祖先祖细胞的发育调节。有趣的是，tdTomato（+）树突状细胞、单核细胞和中性粒细胞的频率与嗜酸性粒细胞中tdTomato（+）的比例无关，这表明嗜酸性粒细胞中Foxp3启动子的活性发生在从一个共同的多能祖细胞分裂后。当这些F-1小鼠进行杂交以实现启动子和报告基因的纯合时，观察到一种新的视觉红色表型在幼崽之间分离。红色在非免疫组织中普遍存在。从这些红小鼠中检测的胸腺细胞显示，基于CD25和CD44差异表达的所有四种未成熟胸腺细胞亚群(CD4（-) CD8(-)）都表达tdTomato。最后，我们展示了Foxp3 Cre重组酶不依赖于tdTomato表达的证据，表明激活的tdTomato报告基因可以种系传播。我们的数据突出了使用B6得出的结论的潜在问题。129 (Cg) foxp3 (tm4 (YFP / Cre)埃尔)/ J小鼠。
Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F-1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato(+) dendritic cells, monocytes and neutrophils did not correlate with the tdTomato(+) fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F-1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4(-) CD8(-)) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6. 129(Cg)-Foxp3(tm4(YFP/Cre)Ayr)/J mice.