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Using Morpholinos to Control Gene Expression

Jon D. Moulton1

1Gene Tools, LLC, Philomath, Oregon

Publication Name: 
Current Protocols in Nucleic Acid Chemistry
Unit Number: 
Unit 4.30
DOI: 
10.1002/0471142700.nc0430s27
Online Posting Date: 
January, 2007
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Jon Moulton

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.

Keywords: Morpholino; antisense; oligo; knockdown; splicing

     
 
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Table of Contents

  • Unit 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 2: Preparation and Verification of Morpholino Stock Solutions

 Materials
  • Lyophilized Morpholino oligonucleotide (Gene Tools)
  • Distilled autoclaved water without DEPC, sterile
  • 0.1 M HCl
  • Glass or polypropylene/polyethylene tubes with labels
  • 65°C water bath
  • 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 3: Delivery of Morpholinos into Cells Using Endo-Porter

 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% serum
  • Fluorescence microscope
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Figures

  • Figure 4.30.1
    Structure of a Morpholino 3-mer.

    View Image
  • Figure 4.30.2
    Comparison of RNase H–dependant, RISC-dependant, and steric blocking oligos.

    View Image
  • Figure 4.30.3
    Targetable regions for translation blocking (A) and splice blocking (B).

    View Image
  • Figure 4.30.4
    Commercially available 3¢-end modifications of Morpholino oligos.

    View Image

Literature Cited

Literature Cited
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    Kinney, R.M., Huang, C.Y., Rose, B.C., Kroeker, A.D., Dreher, T.W., Iversen, P.L., and Stein, D.A. 2005. Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with Morpholino oligomers. J. Virol. 79:5116-5128.
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    Kos, R., Tucker, R.P., Hall, R., Duong, T.D., and Erickson, C.A. 2003. Methods for introducing Morpholinos into the chicken embryo. Dev. Dyn. 226:470-477.
    Lebedeva, I. and Stein, C.A. 2001. Antisense oligonucleotides: Promise and reality. Annu. Rev. Pharmacol. Toxicol. 41:403-419.
    Liu, G., He, J., Zhang, S., Liu, C., Rusckowski, M., and Hnatowich, D.J. 2002a. Cytosine residues influence kidney accumulations of 99mTc-labeled Morpholino oligomers. Antisense Nucleic Acid Drug. Dev. 12:393-398.
    Liu, G., Zhang, S., He, J., Liu, N., Gupta, S., Rusckowski, M., and Hnatowich, D.J. 2002b. The influence of chain length and base sequence on the pharmacokinetic behavior of 99mTc-Morpholinos in mice. Q. J. Nucl. Med. 46:233-243.
    Mang'era, K.O., Liu, G., Yi, W., Zhang, Y., Liu, N., Gupta, S., Rusckowski, M., and Hnatowich, D.J. 2001. Initial investigations of 99mTc-labeled Morpholinos for radiopharmaceutical applications. Eur. J. Nucl. Med. 28:1682-1689.
    Masaki, M., Izumi, M., Oshima, Y., Nakaoka, Y., Kuroda, T., Kimura, R., Sugiyama, S., Terai, K., Kitakaze, M., Yamauchi-Takihara, K., Kawase, I., and Hirota, H. 2005. Smad1 protects cardiomyocytes from ischemia-reperfusion injury. Circulation 111:2752-2759.
    McCaffrey, A.P., Meuse, L., Karimi, M., Contag, C.H., and Kay, M.A. 2003. A potent and specific Morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 38:503-508.
    McClorey, G., Moulton, H.M., Iversen, P.L., Fletcher, S., and Wilton, S.D. 2006. Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther. 13:1373-1381.
    Mellitzer, G., Hallonet, M., Chen, L., and Ang, S.L. 2002. Spatial and temporal ‘knock down’ of gene expression by electroporation of double-stranded RNA and Morpholinos into early postimplantation mouse embryos. Mech. Dev. 118:57-63.
    Monga, S.P., Monga, H.K., Tan, X., Mule, K., Pediaditakis, P., and Michalopoulos, G.K. 2003. Beta-catenin antisense studies in embryonic liver cultures: Role in proliferation, apoptosis, and lineage specification. Gastroenterology 124:202-216.
    Morcos, P.A. 2001. Achieving efficient delivery of Morpholino oligos in cultured cells. Genesis 30:94-102.
    Moulton, H.M. and Moulton, J.D. 2003. Peptide-assisted delivery of steric-blocking antisense oligomers. Curr. Opin. Mol. Ther. 5:123-132.
    Moulton, H.M., Nelson, M.H., Hatlevig, S.A., Reddy, M.T., and Iversen, P.L. 2004. Cellular uptake of antisense Morpholino oligomers conjugated to arginine-rich peptides. Bioconjug. Chem. 15:290-299.
    Nasevicius, A. and Ekker, S.C. 2000. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26:216-220.
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    Neuman, B.W., Stein, D.A., Kroeker, A.D., Paulino, A.D., Moulton, H.M., Iversen, P.L., and Buchmeier, M.J. 2004. Antisense Morpholino-oligomers directed against the 5¢ end of the genome inhibit coronavirus proliferation and growth. J. Virol. 78:5891-5899.
    Neuman, B.W., Stein, D.A., Kroeker, A.D., Churchill, M.J., Kim, A.M., Kuhn, P., Dawson, P., Moulton, H.M., Bestwick, R.K., Iversen, P.L., and Buchmeier, M.J. 2005. Inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense Morpholino oligomers. J. Virol. 79:9665-9676.
    Nutt, S.L., Bronchain, O.J., Hartley, K.O., and Amaya, E. 2001. Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis. Genesis 30:110-113.
    Partridge, M., Vincent, A., Matthews, P., Puma, J., Stein, D., and Summerton, J. 1996. A simple method for delivering Morpholino antisense oligos into the cytoplasm of cells. Antisense Nucleic Acid Drug Dev. 6:169-175.
    Prasadan, K., Daume, E., Preuett, B., Spilde, T., Bhatia, A., Kobayashi, H., Hembree, M., Manna, P., and Gittes, G.K. 2002. Glucagon is required for early insulin-positive differentiation in the developing mouse pancreas. Diabetes 51:3229-3236.
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    Sazani, P., Gemignani, F., Kang, S.H., Maier, M.A., Manoharan, M., Persmark, M., Bortner, D., and Kole, R. 2002. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat. Biotechnol. 20:1228-1233.
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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 printing, >1500 publications have reported experiments with Morpholino oligos in a broad range of systems. Citations and many abstracts are searchable here.

    http://p196.ezboard.com/bmorpholinos

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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Author Notes

Jon Moulton
April 23, 2009

A more up-to-date version of this chapter is available through Current Protocols in Molecular Biology, including an additional protocol for microinjection of Morpholino oligos into embryos:

http://currentprotocols.com/protocol/mb2608

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