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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


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

PDF or HTML at Wiley Online Library

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
PDF or HTML at Wiley Online Library


Basic Protocol 1: Design of a Morpholino Knockdown Experiment

  • 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

  • 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
PDF or HTML at Wiley Online Library



Literature Cited

  Aboul‐ela, F., Koh, D., Tinoco, I. Jr., and Martin, F.H. 1985. Base‐base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T). Nucl. Acids Res. 13:4811‐4824. doi: 10.1093/nar/13.13.4811.
  Alonso, M., Stein, D.A., Thomann, E., Moulton, H.M., Leong, J.C., Iversen, P., and Mourich, D.V. 2005. Inhibition of infectious haematopoietic necrosis virus in cell cultures with eptide‐conjugated morpholino oligomers. J. Fish Dis. 28:399‐410. doi: 10.1111/j.1365‐2761.2005.00641.x.
  Arthur, P.K., Claussen, M., Koch, S., Tarbashevich, K., Jahn, O., and Pieler, T. 2009. Participation of Xenopus ELR‐type proteins in vegetal mRNA localization during oogenesis. J. Biol. Chem. 284:19982‐19992. doi: 10.1074/jbc.M109.009928.
  Bhadra, J., Pattanayak, S., and Sinha, S. 2015. Synthesis of morpholino monomers, chlorophosphoramidate monomers, and solid‐phase synthesis of short morpholino oligomers. Curr. Protoc. Nucleic Acid. Chem. 62:4.65.1‐4.65.26. doi:10.1002/0471142700.nc0465s62.
  Blum, M., De Robertis, E.M., Wallingford, J.B., and Niehrs, C. 2015. Morpholinos: Antisense and sensibility. Dev. Cell. 35:145‐149. doi: 10.1016/j.devcel.2015.09.017.
  Bruno, I.G., Jin, W., and Cote, G.J. 2004. Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements. Hum. Mol. Genet. 13:2409‐2420. doi: 10.1093/hmg/ddh272.
  Cerda, G.A., Thomas, J.E., Allende, M.L., Karlstrom, R.O., and Palma, V. 2006. Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods 39:207‐211. doi: 10.1016/j.ymeth.2005.12.009.
  Cheong, C. and Moore, P.B. 1992. Solution structure of an unusually stable RNA tetraplex containing G‐ and U‐quartet structures. Biochemistry 31:8406‐8414. doi: 10.1021/bi00151a003.
  Choi, W.Y., Giraldez, A.J., and Schier, A.F. 2007. Target protectors reveal dampening and balancing of nodal agonist and antagonist by miR‐430. Science 318:271‐274. doi: 10.1126/science.1147535
  Chu, T.W., Zhang, R., Yang, J., Chao, M.P., Shami, P.J., and Kopeček, J. 2015. A two‐step pretargeted nanotherapy for CD20 crosslinking may achieve superior anti‐lymphoma efficacy to rituximab. Theranostics 5:834‐846. doi:10.7150/thno.12040.
  Deas, T.S., Binduga‐Gajewska, I., Tilgner, M., Ren, P., Stein, D.A., Moulton, H.M., Iversen, P.L., Kauffman, E.B., Kramer, L.D., and Shi, P.Y. 2005. Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J. Virol. 79:4599‐4609. doi: 10.1128/JVI.79.8.4599‐4609.2005.
  Draper, B.W., Morcos, P.A., and Kimmel, C.B. 2001. Inhibition of zebrafish fgf8 pre‐mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis 30:154‐156. doi: 10.1002/gene.1053.
  Ekker, S.C. 2000. Morphants: A new systematic vertebrate functional genomics approach. Yeast 17:302‐306. doi: 10.1002/1097‐0061(200012)17:4%3c302::AID‐YEA53%3e3.0.CO;2‐.
  Ekker, S.C. and Larson, J.D. 2001. Morphant technology in model developmental systems. Genesis 30:89‐93. doi: 10.1002/gene.1038.
  Enterlein, S., Warfield, K.L., Swenson, D.L., Stein, D.A., Smith, J.L., Gamble, C.S., Kroeker, A.D., Iversen, P.L., Bavari, S., and Muhlberger, E. 2006. VP35 knockdown inhibits ebola virus amplification and protects against lethal infection in mice. Antimicrob. Agents Chemother. 50:984‐993. doi: 10.1128/AAC.50.3.984‐993.2006.
  Ferguson, D.P., Dangott, J.J., and Lightfoot, J.T. 2014. Lessons learned from vivo‐morpholinos: How to avoid vivo‐morpholino toxicity. BioTechniques 56:251‐256.
  FDA. 2016. FDA grants accelerated approval to first drug for Duchenne muscular dystrophy. Press release. Available at doi: 10.2144/000114167.
  Geller, B.L., Deere, J., Tilley, L., and Iversen, P.L. 2005. Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis. J. Antimicrob. Chemother. 55:983‐988. doi: 10.1093/jac/dki129.
  Giles, R.V., Spiller, D.G., Clark, R.E., and Tidd, D.M. 1999. Antisense morpholino oligonucleotide analog induces missplicing of c‐myc mRNA. Antisense Nucl. Acid Drug Dev. 9:213‐220. doi: 10.1089/oli.1.1999.9.213.
  Hammond, S.M., Hazell, G., Shabanpoor, F., Saleh, A.F., Bowerman, M., Sleigh, J.N., Meijboom, K.E., Zhou, H., Muntoni, F., Talbot, K., Gait, M.J., and Wood, M.J. 2016. Systemic peptide‐mediated oligonucleotide therapy improves long‐term survival in spinal muscular atrophy. Proc. Natl. Acad. Sci. U.S.A. 113:10962‐10967. doi: 10.1073/pnas.1605731113.
  Hayashi, Y., Horibata, Y., Sakaguchi, K., Okino, N., and Ito, M. 2005. A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates. Anal. Biochem. 345:181‐186. doi: 10.1016/j.ab.2005.05.029.
  He, J., Liu, G., Zhang, S., Rusckowski, M., and Hnatowich, D.J. 2003. Pharmacokinetics in mice of four oligomer‐conjugated polymers for amplification targeting. Cancer Biother. Radiopharm. 18:941‐947. doi: 10.1089/108497803322702905.
  He, J., Liu, G., Gupta, S., Zhang, Y., Rusckowski, M., and Hnatowich, D.J. 2004. Amplification targeting: A modified pretargeting approach with potential for signal amplification‐proof of a concept. J. Nucl. Med. 45:1087‐1095.
  Heasman, J., Kofron, M., and Wylie, C. 2000. Beta‐catenin signaling activity dissected in the early Xenopus embryo: A novel antisense approach. Dev. Biol. 222:124‐134. doi: 10.1006/dbio.2000.9720.
  Henderson, R.E., Kirkegaard, L.H., and Leonard, N.J. 1973. Reaction of diethylpyrocarbonate with nucleic acid components. Adenosine‐containing nucleotides and dinucleoside phosphates. Biochim. Biophys. Acta 294:356‐364. doi: 10.1016/0005‐2787(73)90090‐7.
  Howard, M.T., Gesteland, R.F., and Atkins, J.F. 2004. Efficient stimulation of site‐specific ribosome frameshifting by antisense oligonucleotides. RNA 10:1653‐1661. doi: 10.1261/rna.7810204.
  Hudziak, R.M., Barofsky, E., Barofsky, D.F., Weller, D.L., Huang, S.B., and Weller, D.D. 1996. Resistance of morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev. 6:267‐272. doi: 10.1089/oli.1.1996.6.267.
  Jubin, R. 2005. Optimizing electroporation conditions for intracellular delivery of morpholino antisense oligonucleotides directed against the hepatitis C virus internal ribosome entry site. Methods Mol. Med. 106:309‐322. doi: 10.1385/1‐59259‐854‐4:309.
  Jubin, R., Vantuno, N.E., Kieft, J.S., Murray, M.G., Doudna, J.A., Lau, J.Y., and Baroudy, B.M. 2000. Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding. J. Virol. 74:10430‐10437. doi: 10.1128/JVI.74.22.10430‐10437.2000.
  Kang, H., Chou, P.J., Johnson, W.C. Jr., Weller, D., Huang, S.B., and Summerton, J.E. 1992. Stacking interactions of ApA analogues with modified backbones. Biopolymers 32:1351‐1363. doi: 10.1002/bip.360321009.
  Khokha, M.K., Chung, C., Bustamante, E.L., Gaw, L.W., Trott, K.A., Yeh, J., Lim, N., Lin, J.C., Taverner, N., Amaya, E., Papalopulu, N., Smith, J.C., Zorn, A.M., Harland, R.M., and Grammer, T.C. 2002. Techniques and probes for the study of Xenopus tropicalis development. Dev. Dyn. 225:499‐510. doi: 10.1002/dvdy.10184.
  Kimmel, C.B. and Law, R.D. 1985a. Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer. Dev. Biol. 108:86‐93. doi: 10.1016/0012‐1606(85)90011‐9.
  Kimmel, C.B. and Law, R.D. 1985b. Cell lineage of zebrafish blastomeres. III. Clonal analyses of the blastula and gastrula stages. Dev. Biol. 108:94‐101. doi: 10.1016/0012‐1606(85)90012‐0.
  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. doi: 10.1128/JVI.79.8.5116‐5128.2005.
  Kloosterman, W.P., Wienholds, E., Ketting, R.F., and Plasterk, R.H. 2004. Substrate requirements for let‐7 function in the developing zebrafish embryo. Nucl. Acids Res. 32:6284‐6291. doi: 10.1093/nar/gkh968.
  Kok, F.O., Shin, M., Ni, C‐W., Gupta, A., Grosse, A.S., van Impel, A., Kirchmaier, B.C., Peterson‐Maduro, J., Kourkoulis, G., Male, I., DeSantis, D.F., Sheppard‐Tindell, S., Ebarasi, L., Betsholtz, C., Schulte‐Merker, S., Wolfe, S.A., and Lawson, N.D. 2015. Reverse genetic screening reveals poor correlation between morpholino‐induced and mutant phenotypes in zebrafish. Dev Cell. 32:97‐108. doi: 10.1016/j.devcel.2014.11.018.
  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. doi: 10.1002/dvdy.10254.
  Kumar, V., Maurya, V.K., Joshi, A., Meeran, S.M., and Jha, R.K. 2015. Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF‐B1 in mice. Biol. Reprod. 92:109. doi: 10.1095/biolreprod.114.122838.
  Lebedeva, I. and Stein, C.A. 2001. Antisense oligonucleotides: Promise and reality. Annu. Rev. Pharmacol. Toxicol. 41:403‐419. doi: 10.1146/annurev.pharmtox.41.1.403.
  Li, Y.F. and Morcos, P.A. 2008. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo. Bioconjug Chem. 19:1464‐1470. doi: 10.1021/bc8001437.
  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. doi: 10.1089/108729002321082465.
  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. doi: 10.1007/s002590100637.
  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. doi: 10.1161/CIRCULATIONAHA.104.490946.
  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. doi: 10.1053/jhep.2003.50330.
  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. doi: 10.1038/
  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. doi: 10.1016/S0925‐4773(02)00191‐0.
  Mendell, J., Rodino‐Klapac, L.R., Sahenk, Z., Roush, K., Bird, L., Lowes, L.P., Alfano, L., Gomez, A.M., Lewis, S., Kota, J., Malik, V., Shontz, K., Walker, C.M., Flanigan, K.M., Kean, J.R., Allen, H.D., Shilling, C., Melia, K.R., Sazani, P., Saoud, J.B., and Kaye, E.M., 2013. The Eteplirsen Study Group. Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann. Neurol. 74:637‐647. doi: 10.1002/ana.23982.
  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. doi: 10.1053/gast.2003.50000.
  Morcos, P.A. 2001. Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 30:94‐102. doi: 10.1002/gene.1039.
  Morcos, P.A. 2007. Achieving targeted and quantifiable alteration of mRNA splicing with morpholino oligos. Biochem. Biophys. Res. Commun. 358:521‐527. doi: 10.1016/j.bbrc.2007.04.172.
  Morcos, P.A, Li Y.F., and Jiang S. 2008. Vivo‐Morpholinos: A non‐peptide transporter delivers morpholinos into a wide array of mouse tissues. BioTechniques. 45:616‐626. doi: 10.2144/000113005.
  Morcos, P.A., Vincent, A.C., and Moulton, J.D. 2015. Gene editing versus Morphants. Zebrafish 12:319. doi: 10.1089/zeb.2015.1114.
  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. and Moulton, J.D. 2010. Morpholinos and their peptide conjugates: Therapeutic promise and challenge for duchenne muscular dystrophy. Biochim. Biophys. Acta. 2010:2296‐2303. doi: 10.1016/j.bbamem.2010.02.012.
  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. doi: 10.1021/bc034221g.
  Nasevicius, A. and Ekker, S.C. 2000. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26:216‐220. doi: 10.1038/79951.
  Nelson, M.H., Stein, D.A., Kroeker, A.D., Hatlevig, S.A., Iversen, P.L., and Moulton, H.M. 2005. Arginine‐rich peptide conjugation to morpholino oligomers: Effects on antisense activity and specificity. Bioconjug. Chem. 16:959‐966. doi: 10.1021/bc0501045.
  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. doi: 10.1128/JVI.78.11.5891‐5899.2004.
  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. doi: 10.1128/JVI.79.15.9665‐9676.2005.
  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. doi: 10.1002/gene.1042.
  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. doi: 10.1089/oli.1.1996.6.169.
  Pérez, B., Rincón, A., Jorge‐Finnigan, A., Richard, E., Merinero, B., Ugarte, M., and Desviat, L.R. 2009. Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria). Hum. Mutat. 30:1676‐1682. doi: 10.1002/humu.21118.
  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. doi: 10.2337/diabetes.51.11.3229.
  Reissner, K.J., Sartor, G.C., Vazey, E.M., Dunn, T.E., Aston‐Jones, G., and Kalivas, P.W. 2012. Use of vivo‐morpholinos for control of protein expression in the adult rat brain. J. Neurosci. Meth. 203:354‐360. doi: 10.1016/j.jneumeth.2011.10.009.
  Rossi, A., Kontarakis, Z., Gerri, C., Nolte, H., Hölper, S., Krüger, M., and Stainier, D.Y.R. 2015. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524:230‐233. doi: 10.1038/nature14580.
  Sazani, P., Kang, S.H., Maier, M.A., Wei, C., Dillman, J., Summerton, J., Manoharan, M., and Kole, R. 2001. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucl. Acids Res. 29:3965‐3974. doi:10.1093/nar/29.19.3965.
  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. doi: 10.1038/nbt759.
  Scacheri, P.C., Rozenblatt‐Rosen, O., Caplen, N.J., Wolfsberg, T.G., Umayam, L., Lee, J.C., Hughes, C.M., Shanmugam, K.S., Bhattacharjee, A., Meyerson, M., and Collins, F.S. 2004. Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 101:1892‐1897. doi: 10.1073/pnas.0308698100.
  Schmajuk, G., Sierakowska, H., and Kole, R. 1999. Antisense oligonucleotides with different back bones. Modification of splicing pathways and efficacy of uptake. J. Biol. Chem. 274:21783‐21789. doi: 10.1074/jbc.274.31.21783.
  Shabanpoor, F., McClorey, G., Saleh, A.F., Järver, P., Wood, M.J.A., and Gait, M.J. 2015. Bi‐specific splice‐switching PMO oligonucleotides conjugated via a single peptide active in a mouse model of Duchenne muscular dystrophy. Nucl. Acids Res. 43:29‐39. doi: 10.1093/nar/gku1256.
  Stein, D., Foster, E., Huang, S.B., Weller, D., and Summerton, J. 1997. A specificity comparison of four antisense types: Morpholino, 2′‐ O‐methyl RNA, DNA, and phosphorothioate DNA. Antisense Nucleic Acid Drug Dev. 7:151‐157. doi: 10.1089/oli.1.1997.7.151.
  Stein, D.A., Skilling, D.E., Iversen, P.L., and Smith, A.W. 2001. Inhibition of Vesivirus infections in mammalian tissue culture with antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev. 11:317‐325. doi: 10.1089/108729001753231696.
  Stirchak, E.P., Summerton, J.E., and Weller, D.D. 1989. Uncharged stereoregular nucleic acid analogs: 2. Morpholino nucleoside oligomers with carbamate internucleoside linkages. Nucl. Acids Res. 17:6129‐6141. doi: 10.1093/nar/17.15.6129.
  Summerton, J. 1999. Morpholino antisense oligomers: The case for an RNase H‐independent structural type. Biochim. Biophys. Acta 1489:141‐158. doi: 10.1016/S0167‐4781(99)00150‐5.
  Summerton, J. 2004. Morpholinos and PNAs compared. Lett. Pept. Sci. 10:215‐236. doi: 10.1007/s10989‐004‐4913‐y.
  Summerton, J. 2005. Endo‐Porter: A novel reagent for safe, effective delivery of substances into cells. Ann. N.Y. Acad. Sci. 1058:1‐14. doi: 10.1196/annals.1359.012.
  Summerton, J. 2016. History and properties of morpholino antisense oligos. J. Drug Discov. Develop. Deliv. 3:1019.
  Summerton, J. and Weller, D. 1997. Morpholino antisense oligomers: Design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 7:187‐195. doi: 10.1089/oli.1.1997.7.187.
  Suwanmanee, T., Sierakowska, H., Fucharoen, S., and Kole, R. 2002. Repair of a splicing defect in erythroid cells from patients with beta‐thalassemia/HbE disorder. Mol. Ther. 6:718‐726. doi: 10.1006/mthe.2002.0805.
  Takahashi, M., Sato, K., Nomura, T., and Osumi, N. 2002. Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70:155‐162. doi: 10.1046/j.1432‐0436.2002.700405.x.
  Thummel, R., Bai, S., Sarras, M.P. Jr, Song, P., McDermott, J., Brewer, J., Perry, M., Zhang, X., Hyde, D.R., and Godwin, A.R. 2006. Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb. Dev. Dyn. 235:336‐346. doi: 10.1002/dvdy.20630.
  Tidd, D.M., Giles, R.V., Broughton, C.M., and Clark, R.E. 2001. Expression of c‐myc is not critical for cell proliferation in established human leukemia lines. BMC Mol. Biol. 2:13. doi: 10.1186/1471‐2199‐2‐13.
  Tripathi, S., Pohl, M.O., Zhou, Y., Rodriguez‐Frandsen, A., Wang, G., Stein, D.A., Moulton, H.M., DeJesus, P., Che, J., Mulder, L.C.F., Yángüez, E., Andenmatten, D., Pache, L., Manicassamy, B., Albrecht, R.A., Gonzalez, M.G., Nguyen, Q., Brass, A., Elledge, S., White, M., Shapira, S., Hacohen, N., Karlas, A., Meyer, T.F., Shales, M., Gatorano, A., Johnson, J.R., Jang, G., Johnson, T., Verschueren, E., Sanders, D., Krogan, N., Shaw, M., König, R., Stertz, S., García‐Sastre, A., and Chanda, S.K. 2015. Meta‐ and orthogonal integration of influenza “OMICs” data defines a role for UBR4 in virus budding. Cell Host Microbe 18:723‐735. doi: 10.1016/j.chom.2015.11.002.
  Tucker, R.P. 2004. Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development. Int. J. Biochem. Cell Biol. 36:1135‐1139. doi: 10.1016/j.biocel.2004.01.010.
  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.
  Wada, T., Hara, M., Taneda, T., Qingfu, C., Takata, R., Moro, K., Takeda, K., Kishimoto, T., and Handa, H. 2012. Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation. Nucleic Acids Res. 40:e173. doi: 10.1093/nar/gks765.
  Warfield, K.L., Swenson, D.L., Olinger, G.G., Nichols, D.K., Pratt, W.D., Blouch, R., Stein, D.A., Aman, M.J., Iversen, P.L., and Bavari, S. 2006. Gene‐specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers. PLoS Pathog. 2:e1. doi: 10.1371/journal.ppat.0020001.
  Wu, B., Li, Y., Morcos, PA., Doran, T.J., Lu, P., and Lu, Q.L. 2009. Octa‐guanidine morpholino restores dystrophin expression in cardiac and skeletal muscles and ameliorates pathology in dystrophic mdx mice. Mol. Ther. 17:864‐871. doi: 10.1038/mt.2009.38.
  Yen, L., Svendsen, J., Lee, J.S., Gray, J.T., Magnier, M., Baba, T., D'Amato, R.J., and Mulligan, R.C. 2004. Exogenous control of mammalian gene expression through modulation of RNA self‐cleavage. Nature 431:471‐476. doi: 10.1038/nature02844.
  Youngblood, D.S., Hatlevig, S.A., Hassinger, J.N., Iversen, P.L., and Moulton, H.M. 2007. Stability of cell‐penetrating Peptide‐morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 18:50‐60. doi: 10.1021/bc060138s.
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
  Commercial source for Morpholinos.
  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.
  Zebrafish Information Network. References related to Morpholino use in zebrafish are searchable in an annotated database.‐bin/webdriver?MIval=aa‐newmrkrselect.apg
  Annotated database of zebrafish Morpholino sequences by gene name.
  Sarepta Therapeutics, Inc., Morpholino therapeutics company.
PDF or HTML at Wiley Online Library