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A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice

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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Figure 1: Silencing of FGFR2 exon IIIb.
Figure 2: Cloning strategies for constructing the original fluorescence reporters, Gint and Rint.
Figure 3: Primers and vector used for screening targeted insertion.
Figure 4: Imaging exon IIIb silencing in live animals.
Figure 5: Imaging exon IIIb silencing in whole organs.
Figure 6: Microscopic imaging of exon IIIb silencing.

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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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Correspondence to Mariano A Garcia-Blanco.

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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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