Generation of a Gal4-dependent gene recombination and illuminating mouse.
Generation of a Gal4-dependent gene recombination and illuminating mouse.
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DOI：
10.1538/expanim.21-0202
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发表时间：
2022-08-05
影响因子：
2.4
通讯作者：
Takahashi, Satoru
中科院分区：
文献类型：
--
作者：
Yoshihara, Masaharu;Nishino, Teppei;Sambe, Naoto;Nayakama, Takahiro;Radtke, Freddy;Mizuno, Seiya;Takahashi, Satoru
细胞标记技术，包括Cre/loxP系统，是发育生物学的有力工具。尽管传统的Cre/loxP系统已被广泛用于标记特定基因的表达，但由于技术上的困难，它较少用于标记蛋白质-蛋白质相互作用。在本研究中，我们建立了一个新的gal4依赖性转基因报告小鼠系，表达Cre重组酶和近红外荧光蛋白miRFP670。为了检验这种新产生的转基因小鼠系是否适用于蛋白-蛋白相互作用的标记，我们使用了先前报道的表达Notch1受体的转基因小鼠系，其胞内结构域被酵母转录因子Gal4取代。人工Notch1受体与内源性Notch1配体结合后，将Gal4从细胞膜上切割出来，诱导Cre重组酶和miRFP670的表达。事实上，我们在小鼠胚胎（胚胎期14.5天）中观察到了miRFP670信号。此外，我们通过另一种转基因小鼠系检测了Cre介导重组后表达dsRed的Cre重组酶是否具有功能。我们在小肠上皮细胞中观察到dsRed信号，其中Notch1信号被认为参与了隐窝干细胞的维持，这表明我们的Cre重组酶是功能性的。由于我们新生成的小鼠系只需要Gal4的功能，因此它可以用于标记体内几种类型的分子活动。
Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a near-infrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.
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影响因子：
29.4
作者：
Pellegrinet L;Rodilla V;Liu Z;Chen S;Koch U;Espinosa L;Kaestner KH;Kopan R;Lewis J;Radtke F
通讯作者：
Radtke F
影响因子：
46.9
作者：
Szymczak, AL;Workman, CJ;Vignali, DAA
通讯作者：
Vignali, DAA
影响因子：
64.5
作者：
Kopan R;Ilagan MX
通讯作者：
Ilagan MX
影响因子：
1.5
作者：
Smith, Emma;Claudinot, Stephanie;Radtke, Freddy
通讯作者：
Radtke, Freddy
影响因子：
8.6
作者：
Shemetov AA;Oliinyk OS;Verkhusha VV
通讯作者：
Verkhusha VV