Wiley Interscience has posted the entire Genesis Morpholino special issue on the web.

Gene Tools is on Twitter, posting Morpholino citations as we find them:
http://twitter.com/#!/GeneTools

Jon Moulton is posting notes on some Morpholino papers in his blog:
http://www.myspace.com/127542408/blog

There is a Morpholino discussion group on Nature Networks.

Sections below:

• Controls & calibration
• Morpholino techniques in general
• Zebrafish
• Xenopus
• Chick
• Mouse
• Ascidian
• Lamprey
• Leech
• Nematode
• Snail
• Plants
• Bacteria
• Quantitation
• Visualization (ISH)
• Unusual Applications
• Databases

Review of Morpholino controls, recommended with enthusiasm!
Eisen JS, Smith JC. Controlling morpholino experiments: don't stop making antisense. Development. 2008 May;135(10):1735-43. Epub 2008 Apr 9.

Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC. p53 activation by knockdown technologies. PLoS Genet. 2007 May 25;3(5):e78. Epub 2007 Apr 10.

Regarding knockdowns causing a p53 response, this group shows some mutations have a similar effect:
Danilova N, Kumagai A, Lin J. p53 Upregulation Is a Frequent Response to Deficiency of Cell-Essential Genes. PLoS One. 2010 Dec 31;5(12):e15938.

See the supporting online material of the following citation for methods for characterizing microinjected volume.
Riedel-Kruse IH, Muller C, Oates AC. Synchrony dynamics during initiation, failure, and rescue of the segmentation clock. Science. 2007 Sep 28;317(5846):1911-5. Epub 2007 Aug 16.

For another approach to calibrating injection volume, see the section "Injection setup and calibration" in:
Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC. A primer for morpholino use in zebrafish. Zebrafish. 2009 Mar;6(1):69-77.

Some brief introdutions to Morpholino techniques in developmental biology are posted on SDB CoRE in the Molecular Techniques section.

Review of Morpholino techniques:
Moulton JD, Yan YL. Using morpholinos to control gene expression. Curr Protoc Mol Biol. 2008 Jul;Chapter 26:Unit26.8.

Exon Skipping
Methods and Protocols Series:
Methods in Molecular Biology, Vol. 867
Aartsma-Rus, Annemieke (Ed.)
2012, 440 p. 70 illus., 8 in color.
Humana Press
Hardcover, ISBN 978-1-61779-766-8

Review of Morpholinos for in vivo delivery:
Moulton JD, Jiang S. Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos. Molecules. 2009 Mar 25;14(3):1304-23.

A database of zebrafish Morpholinos is available at Zfin, the Zebrafish Information Network. Look for Morpholino in the pulldown menu.

You can find zebrafish lines with gene-break transposon inserts in zfishbook. These can be reverted to wild-type gene expression with the appropriate splice-modifying Morpholino oligo. For an open-access description, see "In vivo protein trapping produces a functional expression codex of the vertebrate proteome" in Nature Methods.

The Zebrafish Husbandry Association (ZHA) is a professional organization dedicated to promoting the improvement of husbandry standards for usage of the zebrafish (Danio rerio) in scientific research.

This miRNA protocol by Wigard Kloosterman describes using Morpholinos to knockdown miRNA and LNA to detect miRNA in the zebrafish.

Methods for using Morpholinos in zebrafish, from labs with extensive Morpholino experience:
Bill BR, Petzold AM, Clark KJ, Schimmenti LA, Ekker SC. A primer for morpholino use in zebrafish. Zebrafish. 2009 Mar;6(1):69-77.

Here is an open-access special issue of the journal Zebrafish, Zebrafish in education.

Open access review:
Bedell VM, Westcot SE, Ekker SC. Lessons from morpholino-based screening in zebrafish. Briefings in Functional Genomics. 2011;[Epub ahead of print July 10] doi:10.1093/bfgp/elr021

Zebrafish Electroporation

Thummel R, Bailey TJ, Hyde DR. In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina. J Vis Exp. 2011;58:e3603, doi:10.3791/3603

Gene Tools sponsors an open access JoVE video on electroporation in zebrafish ears:
Holmes KE, Wyatt MJ, Shen Y, Thompson DA, Barald KF. Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development. J Vis Exp. 2011;47 http://www.jove.com/details.stp?id=2466 doi: 10.3791/2466.

Cerda GA, Thomas JE, Allende ML, Karlstrom RO, Palma V. Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods. 2006 Jul;39(3):207-11.

Tawk M, Bianco IH, Clarke JD. Focal electroporation in zebrafish embryos and larvae. Methods Mol Biol. 2009;546:145-51.

Zebrafish Microinjection

Mary Mullins of U. Penn. has posted at MBL a zebrafish microinjection through the chorion protocol for a lab course along with an intro for the Morpholino lab.

Video introductions to microinjection:
Rosen JN, Sweeney MF, Mably JD. Microinjection of zebrafish embryos to analyze gene function. J Vis Exp. 2009 Mar 9;(25). pii: 1115. doi: 10.3791/1115. Open Access Video

Yuan S, Sun Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J Vis Exp. 2009 May 7;(27). pii: 1113. doi: 10.3791/1113.

Jochen Wittbrodt wrote "Microinjection of DNA, RNA and tracer dyes into early fish embryos" describing a protocol for injecting zebrafish and Medaka embryos using Eppendorf microinjection gear, posted on Biocompare.

Here is a zebrafish microinjection protocol posted on the Lom Lab Protocols Resource.

The Smith lab at the Gurdon Institute has posted results of their Morpholino screen in Xenopus tropicalis.

Here are video protocols for obtaining and microinjecting Xenopus laevis eggs:
Cross MK, Powers M. Obtaining eggs from Xenopus laevis females. J Vis Exp. 2008 Aug 20;(18). pii: 890. doi: 10.3791/890.

Xenopus Electroporation

Tabler JM, Liu KJ. Electroporation of Craniofacial Mesenchyme. J Vis Exp. 2011;57:e3381, DOI: 10.3791/3381.

Paper on electroporation into the embryonic Xenopus CNS:
Falk J, Drinjakovic J, Leung KM, Dwivedy A, Regan AG, Piper M, Holt CE. Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus. BMC Developmental Biology 2007, 7:107 [epub ahead of print]

Abstract of a paper describing an electroporation protocol optimized for Xenopus tadpole brains:
Bestman JE, Ewald RC, Chiu SL, Cline HT. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc. 2006;1(3):1267-72.

Here is a video protocol for single-cell electroporation in Xenopus tadpole brain:
Hewapathirane DS, Haas K. Single cell electroporation in vivo within the intact developing brain. J Vis Exp. 2008 Jul 11;(17). pii: 705. doi: 10.3791/705.

Xenopus Microinjection

The Harland lab at Berkeley has posted a protocol for microinjecting Morpholinos into Xenopus tropicalis oocytes.

The Khokha Lab at Yale has posted a page of Morpholino handling and microinjection advice that suggests many useful ideas.

Cohen S, Au S, Panté Eacute N. Microinjection of Xenopus laevis oocytes. J Vis Exp. 2009 Feb 23;(24). pii: 1106. doi: 10.3791/1106.

Tanigawa Y, Ikenishi K, Komiya T. A novel method for microinjection into Xenopus eggs and embryos supported in methylcellulose solution. Int J Dev Biol. 2009;53(7):1053-6.

Mimoto MS, Christian JL. Manipulation of Gene Function in Xenopus laevis. Methods Mol Biol. 2011;770:55-75.

M Natalia Vergara, M Valeria Canto-Soler. Rediscovering the chick embryo as a model to study retinal development. Neural Dev. 2012; 7: 22. Published online 2012 June 27. doi: 10.1186/1749-8104-7-22

Carol Erickson's lab at UC Davis posted this protocols page including a chick egg electroporation protocol.

Voiculescu O, Papanayotou C, Stern CD. Spatially and temporally controlled electroporation of early chick embryos. Nat Protoc. 2008;3(3):419-26.

Video protocol for in ovo electroporation of chick:
Blank MC, Chizhikov V, Millen KJ. In ovo electroporations of HH stage 10 chicken embryos. J Vis Exp. 2007;(9):408. Epub 2007 Nov 1.

Video protocol for ex ovo electroporation of older chick:
Luo J, Yan X, Lin J, Rolfs A. Gene Transfer into Older Chicken Embryos by ex ovo Electroporation. J Vis Exp. 2012 Jul 27;(65). pii: 4078. doi: 10.3791/4078.

Weisinger K, Kohl A, Kayam G, Monsonego‐ornan E, Sela‐donenfeld D. Expression of hindbrain boundary markers is regulated by FGF3. Biology Open. 2012;1(2):67–74. doi: 10.1242/bio.2011032
"MO...injected into the hindbrain lumen of 22–25 somite-old embryos (14–15 Hamburger Hamilton stage) by using a pulled glass capillary. Following injection, electroporation was performed...four 45 millisecond pulses of 16–18 volts and pulse intervals of 300 milliseconds"

BTX Genetrodes cite paper by R. Kos et al. for chick electroporation in ovo.

Domínguez-Bendala J, Álvarez-Cubela S, Nieto M, Vargas N, Espino-Grosso P, Sacher VY, Pileggi A, García E, Ricordi C, Inverardi L, Pastori RL. Intracardial Embryonic Delivery of Developmental Modifiers In Utero. Cold Spring Harb Protoc. 2012;doi:10.1101/pdb.prot069427
Injecting mouse embryos using ultrasound guidance; miRNA knockdowns with Vivo-Morpholinos.

Peng H, Wu Y, Zhang Y. Efficient Delivery of DNA and Morpholinos into Mouse Preimplantation Embryos by Electroporation. PLoS ONE. 2012;7(8):e43748. doi:10.1371/journal.pone.0043748

"Here we describe a morpholino-based gene knockdown microinjection protocol to interrogate gene function at the maternal-embryonic transition."
Yao M, Leong D, Hahn-Windgassen A, Foygel K, Jun S, Behr B. Morpholino-mediated gene knockdown in the early mouse embryo. Nature Protocols 2009 DOI: 10.1038/nprot.2009.110

Stein P, Schindler K. Mouse Oocyte Microinjection, Maturation and Ploidy Assessment. J Vis Exp. 2011:(53) doi:10.3791/2851

See also: Vivo-Morpholinos for adult mice.

Veeman MT, Chiba S, Smith WC. Ciona genetics. Methods Mol Biol. 2011;770:401-22.

Tassy O, Dauga D, Daian F, Sobral D, Robin F, Khoueiry P, Salgado D, Fox V, Caillol D, Schiappa R, Laporte B, Rios A, Luxardi G, Kusakabe T, Joly JS, Darras S, Christiaen L, Contensin M, Auger H, Lamy C, Hudson C, Rothbächer U, Gilchrist MJ, Makabe KW, Hotta K, Fujiwara S, Satoh N, Satou Y, Lemaire P. The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program. Genome Res. 2010 Oct;20(10):1459-68. Epub 2010 Jul 20.

Christiaen L, Wagner E, Shi W, Levine M. Microinjection of Morpholino Oligos and RNAs in Sea Squirt (Ciona) Embryos. Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5347

Nikitina N, Bronner-Fraser M, Sauka-Spengler T. Microinjection of RNA and Morpholino Oligos into Lamprey Embryos. 2009. Cold Spring Harb. Protoc.

Weisblat DA, Kuo DH. Microinjection of Helobdella (Leech) Embryos. Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5190

See section 2.4 part D:
Pires da Silva, A. Pristionchus pacificus genetic protocols (July 17, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.114.1, http://www.wormbook.org.

Gharbiah M, Cooley J, Leise EM, Nakamoto A, Rabinowitz JS, Lambert JD, Nagy LM. Pressure Injection of Ilyanassa Snail Embryos. Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5185

Description of a microinjection rig for plant embryos and injection of Morpholino oligos:
Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, Kawano N, Sakakibara T, Namiki S, Itoh K, Otsuka K, Matsuzaki M, Nozaki H, Kuroiwa T, Nakano A, Kanaoka MM, Dresselhaus T, Sasaki N, Higashiyama T. Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature. 2009 Mar 19;458(7236):357-61.

Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, Geller BL. Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture. Antimicrob Agents Chemother. 2006 Aug;50(8):2789-96.

Kamachi Y, Okuda Y, Kondoh H. Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay. Genesis. 2008 Jan 14;46(1):1-7 [Epub ahead of print]

McKeon J, Cho MJ, Khaledi MG. Quantitation of Intracellular Concentration of a Delivered Morpholino Oligomer by Capillary Electrophoresis-Laser- Induced Fluorescence: Correlation with Upregulation of Luciferase Gene Expression. Anal Biochem 2001 Jun 1;293(1):1-7

Protocol for whole mount microRNA-Morpholino in situ hybridization from the Bakkers lab.

Penn AC, Balik A, Greger IH. Steric antisense inhibition of AMPA receptor Q/R editing reveals tight coupling to intronic editing sites and splicing. Nucleic Acids Res. 2012 Nov 20. [Epub ahead of print]

Howard MT, Gesteland RF, Atkins JF. Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides. RNA. 2004 Oct;10(10):1653-1661.

NCBI Entrez Browser for access to GenBank, Medline files and other biotech resources.

The Ensembl Genome Browser for good markups of introns and exons in transcripts, useful for splice blocking Morpholino design.

The Sanger Institute's miRBase for microRNA sequence and structure information.

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