title: Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.
abstract: In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.
authors.affiliation: Department of Biochemistry, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26505, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Murphy
authors.affiliation: Department of Biochemistry, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26505, USA.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Kolandaivelu
authors.affiliation: Department of Biochemistry, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26505, USA.
authors.forename: Visvanathan
authors.initials: V
authors.lastname: Ramamurthy
authors.affiliation: Department of Biochemistry, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26505, USA. pstoilov@hsc.wvu.edu.
authors.forename: Peter
authors.initials: P
authors.lastname: Stoilov
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D004210:Dissection / Q000379:methods; D018274:Electroporation / Q000379:methods*; D005091:Exons; D017930:Genes, Reporter; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012322:RNA Precursors / Q000235:genetics* / Q000302:isolation & purification; D012160:Retina / Q000166:cytology / Q000378:metabolism*
chemical_list: D012322:RNA Precursors
keywords: Alternative splicing; In vivo splicing reporter; Photoreceptor; Retina