Lentiviral vectors to probe and manipulate the Wnt signaling pathway.
Lentiviral vectors to probe and manipulate the Wnt signaling pathway.
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DOI：
10.1371/journal.pone.0009370
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发表时间：
2010-02-23
期刊：
影响因子：
3.7
通讯作者：
Nusse R
中科院分区：
文献类型：
--
作者：
Fuerer C;Nusse R
Wnt信号通路在发育、成体组织动态平衡和干细胞维持中起着关键作用。进一步了解Wnt信号在特定细胞类型中的功能可能有助于慢病毒载体表达Wnt途径的报告或干扰信号的载体。我们已经开发了一套报告Wnt信号活性并区分阴性和未感染细胞的荧光和发光慢病毒载体。这些载体具有7xTcf-EGFP或7xTcf-FFluc(萤火虫荧光素酶)报告盒，紧随其后的是SV40-mCherry或SV40-PuroR(嘌呤霉素N-乙酰转移酶)选择盒。我们还构建了一个载体，通过将PuroR置于7xTcf启动子的控制下，允许以药物为基础选择具有激活的Wnt信号的细胞。最后，我们在SV40PuroR或SV40mCherry骨架中表达了hEF1TCF4(DnTcf4)或结构性活性β-连环素(β-catenin4A)，以构建抑制或激活Wnt信号通路的载体。这些载体将通过Addgene向科学界提供。这些新型慢病毒是探测和操纵Wnt信号的有效工具。在WNT报告病毒中使用选择盒可以区分未感染和无反应的细胞，这是无法选择克隆的实验的重要要求。使用化学发光读数可以对信号进行量化。最后，可选择的载体可以用来抑制或激活Wnt信号通路。总之，这些载体可以在实验环境中探测和调节Wnt信号通路，在这些实验环境中，转基因或基因转移的持久性不能通过非病毒技术实现。
The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling. We have developed a set of fluorescent and luminescent lentiviral vectors that report Wnt signaling activity and discriminate between negative and uninfected cells. These vectors possess a 7xTcf-eGFP or 7xTcf-FFluc (Firefly Luciferase) reporter cassette followed by either an SV40-mCherry or SV40-PuroR (puromycin N-acetyltransferase) selection cassette. We have also constructed a vector that allows drug-based selection of cells with activated Wnt signaling by placing PuroR under the control of the 7xTcf promoter. Lastly, we have expressed dominant-negative Tcf4 (dnTcf4) or constitutively active beta-catenin (β-catenin4A) from the hEF1α promoter in a SV40-PuroR or SV40-mCherry backbone to create vectors that inhibit or activate the Wnt signaling pathway. These vectors will be made available to the scientific community through Addgene. These novel lentiviruses are efficient tools to probe and manipulate Wnt signaling. The use of a selection cassette in Wnt-reporter viruses enables discriminating between uninfected and non-responsive cells, an important requirement for experiments where selection of clones is not possible. The use of a chemiluminescent readout enables quantification of signaling. Finally, selectable vectors can be used to either inhibit or activate the Wnt signaling pathway. Altogether, these vectors can probe and modulate the Wnt signaling pathway in experimental settings where persistence of the transgene or gene transfer cannot be accomplished by non-viral techniques.
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影响因子：
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作者：
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