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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stoilov P, Lin C-H, Damoiseaux R et al (2008) A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators. Proc Natl Acad Sci 105:11218–11223
Orengo JP, Bundman D, Cooper TA (2006) A bichromatic fluorescent reporter for cell-based screens of alternative splicing. Nucleic Acids Res 34:e148
Kuroyanagi H, Ohno G, Sakane H et al (2010) Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans. Nat Protoc 5:1495–1517
Somarelli JA, Schaeffer D, Bosma R et al (2013) Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo. RNA 19:116–127
Newman EA, Muh SJ, Hovhannisyan RH et al (2006) Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays. RNA 12:1129–1141
Takeuchi A, Hosokawa M, Nojima T et al (2010) Splicing reporter mice revealed the evolutionally conserved switching mechanism of tissue-specific alternative exon selection. PLoS One 5:e10946
Ohno G, Hagiwara M, Kuroyanagi H (2008) STAR family RNA-binding protein ASD-2 regulates developmental switching of mutually exclusive alternative splicing in vivo. Genes Dev 22:360–374
Matsuda T, Cepko CL (2004) Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci 101:16–22
Nickerson JM, Goodman P, Chrenek MA et al (2012) Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes. Methods Mol Biol 884:53–69
Magin-Lachmann C, Kotzamanis G, D’Aiuto L et al (2004) In vitro and in vivo delivery of intact BAC DNA—comparison of different methods. J Gene Med 6:195–209
Barnabé-Heider F, Meletis K, Eriksson M et al (2008) Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation. Nat Methods 5:189–196
Young JL, Dean DA (2015) Electroporation-mediated gene delivery (chapter three). Adv Genet 89:49–88
Heller R, Cruz Y, Heller LC et al (2010) Electrically mediated delivery of plasmid DNA to the skin, using a multielectrode array. Hum Gene Ther 21:357–362
Young JL, Barravecchia MS, Dean DA (2014) Electroporation-mediated gene delivery to the lungs. Methods Mol Biol 1121:189–204
Aihara H, Miyazaki J (1998) Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16:867–870
Tevaearai HT, Gazdhar A, Giraud M-N et al (2014) In vivo electroporation-mediated gene delivery to the beating heart. Methods Mol Biol 1121:223–229
De Vry J, MartÃnez-MartÃnez P, Losen M et al (2010) In vivo electroporation of the central nervous system: a non-viral approach for targeted gene delivery. Prog Neurobiol 92:227–244
Heller R, Jaroszeski M, Atkin A et al (1996) In vivo gene electroinjection and expression in rat liver. FEBS Lett 389:225–228
Michaelis M, Sobczak A, Weitzel JM (2014) In vivo microinjection and electroporation of mouse testis. J Vis Exp 23(90)
DeWoody JA, Schupp J, Kenefic L et al. (2004) Universal method for producing ROX-labeled size standards suitable for automated genotyping, BioTechniques 37: 348, 350, 352
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Avon, MA, Three-volume set
Cooper TA (2005) Use of minigene systems to dissect alternative splicing elements. Methods 37:331–340
Acknowledgements
We thank Zachary Wright and Abigail Hayes for advice and assistance in taking pictures. This work was supported by grants from the National Institutes of Health (EY017035), West Virginia Lions, Lions Club International Fund, and an internal grant from West Virginia University. Imaging experiments were performed in the West Virginia University Microscope Imaging Facility, which has been supported by the Mary Babb Randolph Cancer Center and NIH grants P20 RR016440, P30 GM103488, and P20 GM103434.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Murphy, D., Kolandaivelu, S., Ramamurthy, V., Stoilov, P. (2016). Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter. In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 1421. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3591-8_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3591-8_20
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3589-5
Online ISBN: 978-1-4939-3591-8
eBook Packages: Springer Protocols