This behaved strangely when I posted it into this blog - I think it's a MySpace glitch. The video played extremely fast, far too quickly to see the detail. I messed with the slider on the bottom of the screen, paging through manually, then when I clicked the play button again the video played smoothly at the proper speed.
I wrote up a citation list for Morpholinos in chick embryos...and here it is.
Weisinger K, Wilkinson DG, Sela-Donenfeld D. Inhibition of BMPs by follistatin is required for FGF3 expression and segmental patterning of the hindbrain. Dev Biol. 2008 Sep 18. [Epub ahead of print]
Tucker RP, Tran H, Gong Q. Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos. Protoplasma. 2008 Sep 30. [Epub ahead of print]
Papanayotou C, Mey A, Birot AM, Saka Y, Boast S, Smith JC, Samarut J, Stern CD. A Mechanism Regulating the Onset of Sox2 Expression in the Embryonic Neural Plate. PLoS Biol. 2008 Jan 8;6(1):e2 [Epub ahead of print]
Voiculescu O, Papanayotou C, Stern CD. Spatially and temporally controlled electroporation of early chick embryos. Nat Protoc. 2008;3(3):419-26.
Shepard JB, Gliga DA, Morrow AP, Hoffman S, Capehart AA. Versican knock-down compromises chondrogenesis in the embryonic chick limb. Anat Rec (Hoboken). 2008 Jan;291(1):19-27.
Sauka-Spengler T, Barembaum M. Chapter 12 Gain- and Loss-of-Function Approaches in the Chick Embryo. Methods Cell Biol. 2008;87:237-56.
Shigetani Y, Howard S, Guidato S, Furushima K, Abe T, Itasaki N. Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development. Dev Biol. 2008 Jul 15;319(2):346-58. Epub 2008 May 7.
Mende M, Christophorou NA, Streit A. Specific and effective gene knock-down in early chick embryos using morpholinos but not pRFPRNAi vectors. Mech Dev. 2008 Sep 3. [Epub ahead of print]
Matsson H, Eason J, Bookwalter CS, Klar J, Gustavsson P, Sunnegardh J, Enell H, Jonzon A, Vikkula M, Gutierrez I, Riveron JG, Pope M, Bu'lock F, Cox J, Robinson TE, Song F, Brook JD, Marston S, Trybus KM, Dahl N. Alpha-cardiac actin mutations produce atrial septal defects. Hum Mol Genet. 2008 Jan 15;17(2):256-65. Epub 2007 Oct 18
Tavares AT, Andrade S, Silva AC, Belo JA. Cerberus is a feedback inhibitor of Nodal asymmetric signaling in the chick embryo. Development. 2007 Jun;134(11):2051-60.
Hämmerle B, Tejedor FJ. A Novel Function of DELTA-NOTCH Signalling Mediates the Transition from Proliferation to Neurogenesis in Neural Progenitor Cells. PLoS ONE. 2007 Nov 14;2(11):e1169.
Taneyhill LA, Coles EG, Bronner-Fraser M. Snail2 directly represses cadherin6B during epithelial-to-mesenchymal transitions of the neural crest. Development. 2007 Apr;134(8):1481-90. Epub 2007 Mar 7.
Canto-Soler MV, Adler R. Optic cup and lens development requires Pax6 expression in the early optic vesicle during a narrow time window. Dev Biol. 2006 Jun 1;294(1):119-32. Epub 2006 Mar 27
Nakazawa F, Nagai H, Shin M, Sheng G. Negative regulation of primitive hematopoiesis by the FGF signaling pathway. Blood. 2006 Nov 15;108(10):3335-43. Epub 2006 Aug 3
Suzuki-Hirano A, Sato T, Nakamura H. Regulation of isthmic Fgf8 signal by sprouty2. Development. 2005 Jan;132(2):257-65. Epub 2004 Dec 8.
Granata A, Savery D, Hazan J, Cheung BM, Lumsden A, Quaderi NA. Evidence of functional redundancy between MID proteins: implications for the presentation of Opitz syndrome. Dev Biol. 2005 Jan 15;277(2):417-24.
Duong TD, Erickson CA. MMP-2 plays an essential role in producing epithelial-mesenchymal transformations in the avian embryo. Dev Dyn. 2004 Jan;229(1):42-53.
Gerlach-Bank LM, Cleveland AR, Barald KF. DAN directs endolymphatic sac and duct outgrowth in the avian inner ear. Dev Dyn. 2004 Feb; 229(2): 219-30.
Tucker RP. Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development. Int J Biochem Cell Biol. 2004 Jun;36(6):1135-9.
Nakamura H, Katahira T, Sato T, Watanabe Y, Funahashi J. Gain- and loss-of-function in chick embryos by electroporation. Mech Dev. 2004 Sep;121(9):1137-43. Review.
Krull CE. A primer on using in ovo electroporation to analyze gene function. Dev Dyn. 2004 Mar;229(3):433-9.
Sugiyama S, Nakamura H. The role of Grg4 in tectal laminar formation. Development. 2003 Feb 1;130(3):451-462.
Kos R, Tucker RP, Hall R, Duong TD, Erickson CA. Methods for introducing morpholinos into the chicken embryo. Dev Dyn. 2003 Mar;226(3):470-7.
Hall RJ, Erickson CA. ADAM 10: an active metalloprotease expressed during avian epithelial morphogenesis. Dev Biol. 2003 Apr 1;256(1):147-60.
Granata A. Quaderi N.A. The Opitz syndrome gene MID1 is essential for establishing asymmetric gene expression in Hensen's node. Dev Biol. 2003 Jun 15;258(2):397-405.
Sheng G, dos Reis M, Stern CD. Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation. Cell. 2003 Nov 26;115(5):603-13.
Moftah MZ, Downie SA, Bronstein NB, Mezentseva N, Pu J, Maher PA, Newman SA. Ectodermal FGFs induce perinodular inhibition of limb chondrogenesis in vitro and in vivo via FGF receptor 2. Dev Biol. 2002 Sep 15;249(2):270-82.
Tucker RP. Using Antisense Morpholino Oligos to Knockdown Gene Expression in the Chicken Embryo. Acta Histochemica et Cytochemica, 2002, vol. 35, no. 5, pp. 361-366
Tucker RP. Abnormal neural crest cell migration after the in vivo knockdown of tenascin-C expression with morpholino antisense oligonucleotides. Dev Dyn 2001 Sep;222(1):115-9
Kos R, Reedy MV, Johnson RL, Erickson CA. The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. Development 2001 128(8):1467-1479
AVI’s Morpholino peptide conjugates: mdx mouse hearts, SAR studies
Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Li J, Spurney CF, Sali A, Guerron AD, Nagaraju K, Doran T, Lu P, Xiao X, Lu QL. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci U S A. 2008 Sep 19. [Epub ahead of print] http://www.ncbi.nlm.nih.gov/pubmed/18806224
Yin H, Moulton HM, Seow Y, Boyd C, Boutilier J, Iverson P, Wood MJ. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet. 2008 Sep 10. [Epub ahead of print] http://www.ncbi.nlm.nih.gov/pubmed/18784278
Abes R, Moulton HM, Clair P, Yang ST, Abes S, Melikov K, Prevot P, Youngblood DS, Iversen PL, Chernomordik LV, Lebleu B. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies. Nucleic Acids Res. 2008 Sep 16. [Epub ahead of print] http://www.ncbi.nlm.nih.gov/pubmed/18796528
AVI Biopharma's cell-penetrating peptides (CPP) have contributed to a burst of publication. The papers by Wu et al. and Lin et al. describe the use of CPP-Morpholino conjugates (PPMO) to deliver splice-modifying oligos into mice. The mice used, mdx mice, are a mouse model of muscular dystrophy with a mutation in exon 23 introducing a stop codon. The Morpholinos target exon 23, removing the exon and its stop codon from the mature mRNA. Previous experiments with antisense in Duchenne muscular dystrophy (DMD) models have resulted in poor delivery to the cardiomyocytes, but these new studies have shown that the PPMO can deliver antisense from the blood into the cytosol of cardiomyocytes, from whence they freely diffuse into the nucleus (likely through nuclear pores) and interact with complementary sequences of pre-mRNA at a splice junction of exon 23, modifying splicing. This is particularly important for a DMD drug as many people with DMD ultimately die of heart failure; restoration of dystrophin expression in cardiac tissue is a very promising therapeutic path.
The other paper, Abes et al., reports experiments aimed at determining just why it is that the conjugates with the (RXR)4 peptide are so effective at splice blocking. This paper investigates structure-activity relationships for the (RXR)4 peptide-Morpholino conjugates. Modifications of the successful (RXR)4 peptide were tested for their ability to deliver a splice modifying Morpholino oligo into the pLuc 705 HeLa splice reporter cell line, which expresses an easily-quantifiable luciferase reporter gene when the appropriate splice-correcting oligo enters its nucleus. Other peptide sequences can bring more Morpholino into cells, but the conjugates remain mostly trapped in endosomes. Like the other conjugates, some fraction of the (RXR)4 conjugate also remains in the endosome. However it seems a larger fraction of the (RXR)4 conjugate diffuses out of the endosome, enters the nucleus and triggers splice modification than is the case for the other delivery peptide conjugates which were tested. While it seems likely that the (RXR)4 peptide will enter clinical trials in the future, efforts to improve delivery of Morpholino-peptide conjugates will continue. Understanding why (RXR)4 is so effective will guide the design of the next generation of Morpholino-peptide conjugates.
In the chick embryo, two methods are now used for studying the developmental role of genes by loss-of-function approaches: vector-based shRNA and morpholino oligonucleotides. Both have the advantage that loss-of-function can be conducted in a spatially and temporally controlled way by focal electroporation. Here, we compare these two methods. We find that the shRNA expressing vectors pRFPRNAi, even when targeting a non-expressed protein like GFP, cause morphological phenotypes, mis-regulation of non-targeted genes and activation of the p53 pathway. These effects are highly reproducible, appear to be independent of the targeting sequence and are particularly severe at primitive streak and early somite stages. By contrast, morpholinos do not cause these effects. We propose that pRFPRNAi should only be used with considerable caution and that morpholinos are a preferable approach for gene knock-down during early chick development.
Presentation on Morpholino technology for a lay audience: Powerpoint
Here is a Powerpoint presentation targeting a sharp nontechnical audience, intended to explain to the folks in Philomath Oregon just what it is that we do across the street from the Georgia-Pacific mill. I presented to a hometown group of about 60 folks sitting on hay bales during a cool clear night at the Marys Peak Natural Resources Interpretive Center. I was amazed and delighted to hold the interest of this nontechnical audience for about 40 minutes, after which they continued with a half-hour of thoughtful questions after the presentation. I learned something: if you explain tough concepts carefully to a lay audience, they'll get it. Well, perhaps I was just lucky to be presenting to a good crowd in Philomath.
The following three abstracts describe Morpholino experiments toward development of a Duchenne muscular dystrophy treatment and will be presented at the 13th International Congress of the World Muscle Society, Newcastle, September 2008.
Original web page (with all abstracts): http://www.distrofiamuscular.net/abstracts19.htm
3) Dystrophin expression after systemic delivery of morpholino antisense oligonucleotide in mdx mouse: A dose–response analysis
A. Malerba(a), G. Dickson(a) and I. Graham(a)
(a)Centre for Biomedical Sciences, University of London, Royal Holloway, Egham, Surrey, United Kingdom
Duchenne muscular dystrophy (DMD) is the most common congenital muscular disorder. It is caused by mutations that create premature termination of dystrophin translation. One of the most promising approaches for the treatment of DMD is the administration of antisense oligonucleotide (AO). This approach applied to human muscle cells and in animal models of DMD restored the correct reading frame in the dystrophin transcript, thereby producing a shorter but partially functional protein. One of the most widely used AO chemistries is the phosphorodiamidate morpholino oligomer (morpholino). While it has been clearly demonstrated that administration of morpholino to mdx mice allows the skipping of the mutated exon 23, little is known about the pharmacokinetics and the best doses for the administration of this AO. It has been demonstrated that several injections of morpholino induce the expression of dystrophin in more fibres than after a single injection, but no data have been published that compare the administration of the same total dose over a set period of time using different numbers of administrations or different routes. Here we report that multiple injections of a low amount of morpholino show significantly more dystrophin positive fibres in a wide variety of muscle groups 8 weeks after administration than a single high dose with the total amount. Other histological features, such as the cross sectional area, the centronucleation index and the expression of the dystrophin-associated protein complex, showed a significant improvement in mice treated by repeated injection. These results show that it is feasible to restore dystrophin expression with high efficiency by repeated injections of low doses of morpholino.
4) Induced exon skipping in normal and mdx muscle
S. Fletcher(a), J.P. Steinhaus(a), C. Mitrpant(a), P.L. Meloni(a) and S.D. Wilton(a)
(a)Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Perth, Australia
Induced exon skipping to remove or by-pass protein truncating mutations in the dystrophin gene is emerging as a potential therapy for many cases of Duchenne muscular dystrophy. It has been proposed that the compromised sarcolemma of the dystrophic muscle fibres may facilitate uptake of compounds that induce exon skipping. If this were the case, then restoration of some functional dystrophin expression may restrict further oligomer uptake, thereby creating a therapeutic ceiling. We addressed this question by systemically treating normal C57BL/10ScSn mice with the same compound that induced substantial dystrophin exon 23 skipping and restored dystrophin expression in the mdx mouse model of muscular dystrophy (C57BL/10ScSnmdx). Repeated intraperitoneal injections of a phosphorodiamidate morpholino oligomer coupled to a cell penetrating peptide (PMO-P007), were sufficient to induce readily detectable levels of dystrophin gene transcripts missing exon 23 in normal skeletal muscle, as detected by RT-PCR. However, exon 23 skipping could not be detected in the heart until assay conditions were biased towards generation of shorter PCR products, after which 22% exon skipping was apparent in cardiac muscle from treated animals. Detailed protein studies were not possible on the normal dystrophin-positive background, but clearly, the uptake and efficacy of PMO-P007 was not compromised by the normal skeletal muscle sarcolemma. Furthermore, the selective bias that can be achieved to enhance apparent exon skipping during RT-PCR assays was such that we recommend molecular testing should be standardised to facilitate valid comparisons between different laboratories and studies.
5) Restoration of dystrophin expression in mdx mouse by peptide-conjugated antisense oligonucleotide
(1)Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom (2)University of Oxford, Oxford, United Kingdom (3)AVI BioPharma Inc., Corvalis, United States
Duchene muscular dystrophy (DMD) is a severe muscle wasting disease caused by mutations in the dystrophin gene. The efficacy of antisense oligonucleotide (AO)-mediated exon skipping for the restoration of dystrophin has been established in animal models and in DMD patients. However there remain significant limitations to this therapeutic approach due to the lack of effective systemic AO delivery to muscle. Here we investigate systemic muscle-specific AO delivery by testing AOs directly conjugated to cell penetrating peptides (CPPs) or to tissue-specific homing peptides (e.g. muscle-specific peptide, MSP). Morpholino chemistry AOs were directly conjugated to CPPs or to homing peptides and evaluated in mdx mice following systemic delivery. Effective exon skipping and dystrophin expression were induced in body-wide skeletal muscles and in cardiac muscles. This is the first time that the successful body-wide restorations of dystrophin expression even in heart have been achieved at low AO doses. In parallel we also report the discovery and characterization of a novel delivery formulation which facilitates AO uptake in muscle. A series of studies have shown that this delivery formulation enhances the delivery of AOs of different chemistry (e.g. 2-OMeRNA, PNA and morphlino), depends on the activity of specific muscle membrane transporters, and that it induces significant restoration of dystrophin expression in muscle compared with commonly used delivery formulations. In summary, we report data demonstrating the potential of muscle-specific homing peptides, CPPs and novel delivery formulations for the targeted restoration of dystrophin in DMD.
Many techniques, many organisms, lots of practical lab advice all bundled into one shiny new chapter -- cracklin' dry, but of course. If you are thinking of using a Morpholino, this is (IMHO) a good place to start.
Jim Summerton (inventor of Morpholino oligos) calls them Morpholinos and that's how Gene Tools markets them.
After Summerton's departure from AVI, Pat Iversen (who took over R&D at AVI Biopharma when Summerton left) called them PMO (phosphorodiamidate Morpholino oligomer).
Prior to that, AVI was already calling them Neugenes.
Gene Tools customers sometimes use their own acronyms, such as MORF (from the Hnatowich lab) or MO (in fairly common use, standing for Morpholino oligo).
Morpholino = Neugene = PMO = MORF = MO
Then we have the peptide-linked Morpholino oligos, PPMO, which were developed at AVI Biopharma. These were the oligos used for the paper just published by Ryszard Kole's group in collaboration with AVI: Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S, Iversen PL, Kole R. Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice. Mol Ther. 2008 Jun 10. [Epub ahead of print] The PPMO-B used in this paper is a Morpholino with a particular delivery peptide attached. AVI has built and assayed many, many different peptide conjugates seeking good delivery efficacy with minimal toxicity.
My friend and colleague Yongfu Li considered the papers by Paul Wender, who published his group's work with arginine-rich peptides. Wender also reported some experiments with the guanidinium groups on alternative (non-peptide) support structures. Realizing that it is the guanidinium head groups of the arginine amino acids that give the peptide moieties of the PPMOs their efficacy, Yongfu set out to find a commercially useful method for synthesizing eight-tipped scaffolds, called dendrimers, that can be built on Morpholinos and then decorated with a guanidinium at each tip. This presents the guanidiniums in a hemispherical shape. Yongfu predicted that this hemispherical presentation would distort the endosomal membrane very effectively and lead to good delivery. So far, the data is looking good. Morpholinos with these guanidinium-tipped dendrimers are called Vivo-Morpholinos. Yongfu's paper describing the synthesis and initial biological assays of Vivo-Morpholinos is: Li YF, Morcos PA. Design and Synthesis of Dendritic Molecular Transporter that Achieves Efficient in Vivo Delivery of Morpholino Antisense Oligo. Bioconjug Chem. 2008 Jun 20. [Epub ahead of print]
So, we have two general kinds of Morpholino oligos with delivery groups enhancing in-vivo delivery: PPMO (including PPMO-B) from AVI Vivo-Morpholinos from Gene Tools.
I was blessed with a biochem professor who would drop memorable pearls of wisdom like this: "There is no field so simple that we cannot muck it up by introducing more complex nomenclature". Sigh.
