A protocol for imaging alternative splicing regulation in vivo using fluorescence reporters in transgenic mice.
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.
Bonano
Vivian I
VI
Department of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Oltean
Sebastian
S
Garcia-Blanco
Mariano A
MA
D017398:Alternative Splicing; D000818:Animals; D003001:Cloning, Molecular; D015925:Cryopreservation; D020868:Gene Silencing; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis; D008164:Luminescent Proteins / Q000032:analysis; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051497:Receptor, Fibroblast Growth Factor, Type 2; D000097573:Red Fluorescent Protein; C496354:Fgfr2 protein, mouse