Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.
Dynamic High-Sensitivity Quantitation of Procollagen-I by Endogenous CRISPR-Cas9 NanoLuciferase Tagging.
复制标题
DOI：
10.3390/cells9092070
复制
发表时间：
2020-09-10
期刊：
影响因子：
6
通讯作者：
Pickard A
中科院分区：
文献类型：
--
作者：
Calverley BC;Kadler KE;Pickard A
在活细胞中以亚细胞分辨率在时间和空间上定量感兴趣的蛋白质的能力将为研究和药物发现带来新的机会，但仍然是一个主要的技术挑战。在这里，我们描述了使用纳米荧光酶（NLuc）标记的动态、高灵敏度蛋白质定量技术，该技术在显微镜和多孔平台上都是有效的。使用胶原蛋白作为测试蛋白，crispr - cas9介导将nluc（编码nluc）引入Col1a2位点，实现了原胶原- i （PC-I）定量的简化和小型化。选择胶原蛋白是因为临床对其在心血管和肌肉骨骼疾病以及纤维化中的失调感兴趣，这是45%的死亡（包括癌症引起的死亡）的混淆因素。胶原蛋白也是研究蛋白质分泌的理想载体蛋白，因为它的形状和大小不同寻常。然而，在标记胶原的荧光显微镜下，通常需要使用过表达启动子（其淹没了内源性调节机制）来获得良好的信号/噪声比。我们发现，内源性敲入NLuc，结合其高亮度，不需要使用外源性启动子，保留胶原合成的昼夜节律调节和对TGF-β的反应，并使含有前胶原货物的细胞内运输室的延时显微镜成为可能。总之，我们证明了crispr - cas9介导的内源性NLuc标记在定量细胞外、细胞内和亚细胞蛋白水平和定位方面的应用。
The ability to quantitate a protein of interest temporally and spatially at subcellular resolution in living cells would generate new opportunities for research and drug discovery, but remains a major technical challenge. Here, we describe dynamic, high-sensitivity protein quantitation technique using NanoLuciferase (NLuc) tagging, which is effective across microscopy and multiwell platforms. Using collagen as a test protein, the CRISPR-Cas9-mediated introduction of nluc (encoding NLuc) into the Col1a2 locus enabled the simplification and miniaturisation of procollagen-I (PC-I) quantitation. Collagen was chosen because of the clinical interest in its dysregulation in cardiovascular and musculoskeletal disorders, and in fibrosis, which is a confounding factor in 45% of deaths, including those brought about by cancer. Collagen is also the cargo protein of choice for studying protein secretion because of its unusual shape and size. However, the use of overexpression promoters (which drowns out endogenous regulatory mechanisms) is often needed to achieve good signal/noise ratios in fluorescence microscopy of tagged collagen. We show that endogenous knock-in of NLuc, combined with its high brightness, negates the need to use exogenous promoters, preserves the circadian regulation of collagen synthesis and the responsiveness to TGF-β, and enables time-lapse microscopy of intracellular transport compartments containing procollagen cargo. In conclusion, we demonstrate the utility of CRISPR-Cas9-mediated endogenous NLuc tagging to robustly quantitate extracellular, intracellular, and subcellular protein levels and localisation.
登录
查看更多内容
DOI：
10.1093/bioinformatics/btv308
发表时间：
2015-09-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Hodgkins A;Farne A;Perera S;Grego T;Parry-Smith DJ;Skarnes WC;Iyer V
通讯作者：
Iyer V
影响因子：
7.7
作者：
Kalson NS;Lu Y;Taylor SH;Starborg T;Holmes DF;Kadler KE
通讯作者：
Kadler KE
影响因子：
5.6
作者：
HULMES, DJS;KADLER, KE;PROCKOP, DJ
通讯作者：
PROCKOP, DJ
影响因子：
56.9
作者：
Suter, David M.;Molina, Nacho;Naef, Felix
通讯作者：
Naef, Felix
DOI：
10.1096/fj.12-225599
发表时间：
2013-05
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
--
作者：
Heinemeier KM;Schjerling P;Heinemeier J;Magnusson SP;Kjaer M
通讯作者：
Kjaer M