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

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

He tweets about Morpholinos too, mostly new paper citations with PubMed links.

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
• 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.

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.

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

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.

USA Commercial Drug Discovery Customers!
Are you interested in using Morpholinos in zebrafish, or in having another firm do your Morpholino injections for you? Zygogen offers flexible research and drug discovery licenses as well as contract research services. For-Profit Entities in the USA wishing to use Morpholinos in zebrafish are required to obtain a license from Zygogen (www.zygogen.com). For license or contract research information, please call: 404-523-7309 or email: info@zygogen.com.

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

Here is the Zebrafish Genome Wiki.

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.

Zebrafish Electroporation

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.

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.

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

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.

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.

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.

"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

See also: Vivo-Morpholinos for adult mice.

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

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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