Imaging Histone Methylations in Living Animals
Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions. Since histone methylation is dysregulated in various cellular diseases, it has been considered a potential therapeutic target for drug development. Currently there is no simple method available to screen and preclinically evaluate drugs modulating this cellular process, we recently developed two different methods by adopting reporter gene technology to screen drugs and to preclinically evaluate them in living animals. Method detects and quantitatively monitors the level of histone methylations in intact cells, is of a prerequisite to screen small molecules that modulate histone lysine methylation. Here, we describe two independent optical imaging sensors developed to image histone methylations in cells and in living animals. Since we used standard PCR-based cloning strategies to construct different plasmid vectors shown in this chapter, we are not providing any details regarding the construction methods, instead, we focus on detailing various methods used for measuring histone methylation-assisted luciferase quantitation in cells and imaging in living animals.
Key wordsHistone methylation Optical imaging Reporter genes Luciferase Split reporters Protease Degron In vivo imaging
The funding support by National Institutes of Health (NIH grant R01 CA161091 and R21 CA185805 to R.P) and Department of Radiology, Stanford University is gratefully acknowledged. We also thank Dr. Sanjiv Sam Gambhir, Chairman, Department of Radiology, Stanford University, for his constant support. We gratefully acknowledge the use of the SCi3 Core Facility and Canary Center, Stanford University.
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