Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein–protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

中文翻译：

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在 BMAL1-BioID 敲入小鼠中通过生物素喂养有效诱导邻近依赖性标记

邻近依赖性生物素识别 (BioID) 是识别未知蛋白质-蛋白质相互作用的有用方法。很少有报道描述用于体内 BioID 的基因工程敲入小鼠模型。因此，对于生物素给药的正确方法以及适用的组织知之甚少。在这里，我们建立了脑和肌肉 ARNT-Like 1 (BMAL1) 和具有 R118G 突变的 BirA 生物素连接酶 (BirA*) 的 BioID 敲入小鼠模型。BMAL1-BioID 小鼠模型用于研究饲喂生物素饮食对几种组织中蛋白质生物素化的影响。BMAL1-BirA* 融合蛋白保留了 BMAL1 的正确细胞内定位并与 HEK293T 细胞中的 CLOCK 蛋白结合。小鼠胚胎成纤维细胞中的生物素标记测定揭示了在敲入小鼠细胞中表达的 BMAL1-BirA* 的蛋白质生物素化活性，这取决于生物素的补充。最后，喂食 0.5% 生物素饮食 7 天可诱导 BMAL1-BioID 小鼠大脑、心脏、睾丸和肝脏中的蛋白质生物素化，而不会对精子发生产生不利影响。在肾脏中，生物素饮食增加了 BMAL1-BioID 和对照小鼠的生物素化蛋白水平，表明存在内源性生物素化活性。这些结果为优化体内 BioID 程序提供了有价值的信息。生物素饮食增加了 BMAL1-BioID 和对照小鼠的生物素化蛋白水平，表明存在内源性生物素化活性。这些结果为优化体内 BioID 程序提供了有价值的信息。生物素饮食增加了 BMAL1-BioID 和对照小鼠的生物素化蛋白水平，表明存在内源性生物素化活性。这些结果为优化体内 BioID 程序提供了有价值的信息。