Abstract
Cell-based splicing of minigenes is used extensively in the analysis of alternative splicing events. In particular, such assays are critical for identifying or confirming the in vivo relevance of cis- and trans-acting factors in the regulation of particular splicing patterns. Here we provide detailed information on the methods specific to the cell-based analysis of minigene splicing. In addition, we discuss some of the theoretical considerations that must be given to the design of the minigene and subsequent experimental conditions.
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References
Rothrock C, Cannon B, Hahm B et al (2003) A conserved signal-responsive sequence mediates activation-induced alternative splicing of CD45. Mol Cell 12(5):1317–1324
Xie J, Black DL (2001) A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels. Nature 410(6831):936–939
Zhu J, Mayeda A, Krainer AR (2001) Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. Mol Cell 8(6):1351–1361
Topp JD, Jackson J, Melton AA et al (2008) A cell-based screen for splicing regulators identifies hnRNP LL as a distinct signal-induced repressor of CD45 variable exon 4. RNA 14(10):2038–2049
Stoilov P, Lin CH, Damoiseau R et al (2008) A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators. Proc Natl Acad Sci U S A 105(32):11218–11223
Warzecha CC, Sato TK, Nabet B et al (2009) ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 33(5):591–601
Kramer MF, Coen DM (2001) Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr Protoc Mol Biol. Chapter 15: Unit 15 1
Struhl K (2001) Subcloning of DNA fragments. Curr Protoc Mol Biol. Chapter 3: Unit 3 16
Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY
Anderson K, Moore MJ (1997) Bimolecular exon ligation by the human spliceosome. Science 276(5319):1712–1716
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Smith, S.A., Lynch, K.W. (2014). Cell-Based Splicing of Minigenes. In: Hertel, K. (eds) Spliceosomal Pre-mRNA Splicing. Methods in Molecular Biology, vol 1126. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-980-2_18
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DOI: https://doi.org/10.1007/978-1-62703-980-2_18
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Publisher Name: Humana Press, Totowa, NJ
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