Abstract
Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.
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Acknowledgments
This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement No. 812968 PERICO and the Biotechnology and Biological Sciences Research Council (BB/R016844/1; BB/T002255/1).
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Silva, B.S.C., Schrader, T.A., Schrader, M., Carmichael, R.E. (2023). Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 2643. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3048-8_18
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DOI: https://doi.org/10.1007/978-1-0716-3048-8_18
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