Using Morpholinos to Control Gene Expression

Jon D. Moulton1, Yi‐Lin Yan2

1 Gene Tools, LLC, Philomath, Oregon, 2 Institute of Neuroscience, University of Oregon, Eugene, Oregon
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.8
DOI:  10.1002/0471142727.mb2608s83
Online Posting Date:  July, 2008
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Abstract

Morpholino oligonucleotides are stable, uncharged, water‐soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read‐through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre‐mRNA, and maturation of miRNA, although they can also inhibit 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. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or 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 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. Curr. Protoc. Mol. Biol. 83:26.8.1‐26.8.29. © 2008 by John Wiley & Sons, Inc.

Keywords: Morpholino; antisense; oligo; knockdown; splicing; zebrafish; Xenopus

     
 
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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
  • Basic Protocol 4: Injection of Morpholinos into Embryos of Fish or Amphibians
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Design of a Morpholino Knockdown Experiment

  Materials
  • Lyophilized Morpholino oligonucleotide (Gene Tools)
  • Distilled autoclaved water without DEPC, sterile
  • 0.1 M HCl (aqueous)
  • 65°C water bath
  • 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
  • Cell cultures in plates or flasks at 80% to 100% confluence
  • 1 mM fluoresceinated dextran (10 kDa; e.g., Invitrogen) or 1 mM fluoresceinated Morpholino stock solution ( protocol 2)
  • Cell culture medium with 10% (v/v) serum
  • 1 mM Endo‐Porter solution (aqueous or DMSO formulation, Gene Tools)
  • Fluorescence microscope with fluorescein filter cube (e.g., with filters for 501.5‐nm excitation and 524.5‐nm emissions)

Basic Protocol 3: Delivery of Morpholinos into Cells Using Endo‐Porter

  Materials
  • Embryo medium (with antibiotic, if desired; see recipe)
  • Agarose (e.g., Amresco)
  • Morpholino stock solution ( protocol 2)
  • 1% (w/v) aqueous phenol red, sodium salt (e.g., Sigma)
  • Embryos: e.g., single‐cell Zebrafish embryos, one to eight cell frog embryos
  • 1.2‐mm o.d., 0.94‐mm i.d. glass capillary tubes (e.g., FHC, Harvard Apparatus, or Sutter Instrument)
  • Needle puller (e.g., Sutter Instrument)
  • 100 × 20−mm transparent plastic dish, sterile
  • 1‐mm wide plastic mold to form linear wedge‐shaped troughs atop the agarose block (Adaptive Science Tools)
  • 1.5‐ml microcentrifuge tubes
  • 0.5‐ to 10‐µl pipet tip for filling microinjection capillaries (e.g., microloaders, Eppendorf)
  • Storage jar with electrode gasket to hold injection needle (World Precision Instruments), optional
  • Microinjection system including:
    • Nitrogen gas and regulator (if required)
    • Pressure injector with foot switch (e.g., Applied Scientific Instrumentation or Eppendorf)
    • Micromanipulator (e.g., Märzhäuser)
    • Microelectrode holder for 1.2‐mm‐o.d. glass, with port (e.g., World Precision Instrument)
  • Dissecting microscope (e.g., Leica S4E)
  • Controlled‐drop borosilicate Pasteur pipet, capable of delivering 22 to 26 drops per ml (e.g., Fisher)
  • Pipet pump with thumbwheel (e.g., Scienceware, Fisher Scientific)
  • Forceps
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Figures

Videos

Literature Cited

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Key References
   Draper et al., 2001. See above.
  First description of splice inhibition 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 inhibition 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 printing, >2000 publications have reported experiments with Morpholino oligos in a broad range of systems. Citations and many abstracts are searchable here.
   http://morpholinos.yuku.com
  Discussion board for Morpholino users.
   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.avibio.com
  AVI BioPharma, Inc., Morpholino therapeutics company.
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