Abstract
Imaging technologies are influencing the way we study regulatory processes in vivo. Several recent reports use fluorescence minigenes to image alternative splicing events in living cells and animals. This type of reporter is being used to generate transgenic mice to visualize splicing regulation in diverse tissues and cell types. In this protocol, we describe how to develop animals that report on alternative splicing and how to assess reporter expression in excised organs and tissue sections. The entire procedure, from making the reporters to imaging organs and tissues in adult transgenic mice, should take approximately 1.5 years. Fluorescence reporters can be used to image many splicing decisions in normal tissues and organs and can be extended to the study of disease states.
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Acknowledgements
We thank the present and past members of Garcia-Blanco lab for their suggestions, especially Todd Albrecht, Andrea Baines, Robert Brazas and Eric Wagner for their early work on the development of the fluorescence reporters. We acknowledge support from grants from the National Cancer Institute (1R33 CA97502) and National Institutes of Health (NIH) (1RO1 GM63090) to M.A.G.-B., and NIH (1RO1 GM63090 supplement) to V.I.B.
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Bonano, V., Oltean, S. & Garcia-Blanco, M. A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice. Nat Protoc 2, 2166–2181 (2007). https://doi.org/10.1038/nprot.2007.292
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DOI: https://doi.org/10.1038/nprot.2007.292
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