Using Morpholinos to Control Gene Expression

Jon D. Moulton1

1 Gene Tools, LLC, Philomath
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.30
DOI:  10.1002/cpnc.21
Online Posting Date:  March, 2017
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Abstract

Morpholino oligonucleotides are stable, uncharged, water‐soluble molecules used to block complementary sequences of RNA, preventing processing, read‐through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre‐mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non‐antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein‐mediated catalysis—nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.

Keywords: Morpholino; antisense; oligo; knockdown; splicing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Design of a Morpholino Knockdown Experiment
  • Basic Protocol 2: Preparation and Verification of Morpholino Stock Solutions
  • Basic Protocol 3: Delivery of Morpholinos Into Cells Using Endo‐Porter
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Design of a Morpholino Knockdown Experiment

  Materials
  • Lyophilized Morpholino oligo (Gene Tools)
  • Distilled autoclaved water without DEPC, sterile
  • 0.1 M HCl
  • Glass or polypropylene/polyethylene tubes with labels
  • Quartz spectrophotometer cell (1‐cm path length)
  • Parafilm
  • Lint‐free lab tissues
  • UV spectrophotometer (or UV colorimeter) capable of measurements at 265 nm
  • Morpholino product information sheet

Basic Protocol 2: Preparation and Verification of Morpholino Stock Solutions

  Materials
  • 1 mM Endo‐Porter solution (aqueous or DMSO formulation; Gene Tools)
  • Cell cultures in plates or flasks at 80% to 100% confluence
  • 1 mM Morpholino stock solution (Gene Tools)
  • 1 mM fluoresceinated dextran, 10 kDa
  • Cell culture medium with 10% or less serum
  • Fluorescence microscope
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Figures

Videos

Literature Cited

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  Tyson‐Capper, A.J. and Europe‐Finner, G.N. 2006. Novel targeting of cyclooxygenase‐2 (COX‐2) pre‐mRNA using antisense morpholino oligonucleotides directed to the 3′ acceptor and 5′ donor splice sites of exon 4: Suppression of COX‐2 activity in human amnion‐derived WISH and myometrial cells. Mol. Pharmacol. 69:796‐804. doi: 10.1124/mol.105.020529.
  van den Born, E., Stein, D.A., Iversen, P.L., and Snijder, E.J. 2005. Antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture. J. Gen. Virol. 86:3081‐3090. doi: 10.1099/vir.0.81158‐0.
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Key References
  Draper et al., 2001. See above.
  First description of splice blocking in a zebrafish, including an analysis of a cryptic splice site.
  Nelson et al., 2005. See above.
  Description of peptide‐Morpholino conjugates now in use for in vivo experiments.
  Summerton, 1999. See above.
  Review article presenting data determining the effective region for targeting translation blocking oligos and presenting a detailed discussion of Morpholino specificity and minimum inhibitory length.
  Summerton and Weller, 1997. See above.
  Structure and early synthetic scheme for morpholino oligos.
Internet Resources
  http://www.gene‐tools.com
  Commercial source for Morpholinos.
  http://pubs.gene‐tools.com
  Morpholino publication database. As of late 2016, >8000 publications have reported experiments with morpholino oligos in a broad range of systems. Citations and many abstracts are searchable here.
  http://www.zfin.org
  Zebrafish Information Network. References related to Morpholino use in zebrafish are searchable in an annotated database.
  http://zfin.org/cgi‐bin/webdriver?MIval=aa‐newmrkrselect.apg
  Annotated database of zebrafish Morpholino sequences by gene name.
  http://www.sarepta.com/
  Sarepta Therapeutics, Inc., Morpholino therapeutics company.
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