Article

Recombination-induced tag exchange to track old and new proteins

Division of Gene Regulation, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2009; 107(1):64-8. DOI: 10.1073/pnas.0911164107
Source: PubMed

ABSTRACT The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.

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Available from: Daniel Gottschling, Jun 09, 2014
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    • "Thereby, RITE can be combined with proteomics methods, genomics methods, or DNA-based highthroughput screens (Verzijlbergen et al. 2010; De Vos et al. 2011; Radman-Livaja et al. 2011; Verzijlbergen et al. 2011). The fluorescent tags in the RITE cassettes can be applied to measure protein dynamics in single cells by live imaging (Verzijlbergen et al. 2010; Hotz et al. 2012; Menendez-Benito et al. 2013) and further expanded toward high-throughput genetic screening. The suite of RITE cassettes enables a flexible design for many applications. "
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    • "Based on existing knowledge and reagents, we developed a dual-color histone labeling system, similar to a previously reported strategy (Verzijlbergen et al. 2010). Using this system, preexisting histones can be precisely distinguished from newly synthesized histones because an irreversible DNA recombination induced the old-to-new histone switch (Tran et al. 2012). "
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    • "In order to track SPB inheritance, we developed an assay to unambiguously distinguish old and new SPBs (Figure 1A). Similar to Verzijlbergen et al. (2010), this assay uses recombinase-dependent exchange of fluorescent tags, allowing the discrimination of proteins synthesized before and after recombination . A cassette containing the mCherry coding sequence flanked by loxP recombination sites and followed by yeGFP was inserted after the SPC42 locus, a core component of the SPB (Donaldson and Kilmartin, 1996). "
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