Morpholin Flashcards
A morpholino, also known as a morpholino oligomer and as a phosphorodiamidate morpholino oligomer (PMO), is type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. The molecular structure has a backbone of methylenemorpholine rings and phosphorodiamidate linkages.
A Brief Introduction to Morpholino Antisense
eg:
The protein envelope surrounding the virus was then reversed, in a process called antisense.
在一个称作反义的过程中，病毒外部的蛋白质包膜被反转。
Morpholino oligos are advanced tools for blocking sites on RNA to obstruct cellular processes. A Morpholino oligo specifically binds to its selected target site to block access of cell components to that target site. This property can be exploited to block translation, block splicing, block miRNAs or their targets, and block ribozyme activity.
Antisense therapy
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular geneis known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA.
This synthesized nucleic acid is termed an “antisense” oligonucleotide (ASO) because its base sequence is complementary to the gene’s messenger RNA (mRNA), which is called the “sense” sequence (so that a sense segment of mRNA “ 5’-AAGGUC-3’ “ would be blocked by the anti-sense mRNA segment “ 3’-UUCCAG-5’ “).
Antisense oligonucleotides have been researched as potential drugs for diseases such as cancers (including lung cancer,colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, Amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy and diseases such as asthma, arthritis and pouchitis with an inflammatorycomponent. As of 2014 two antisense drugs have been approved by the U.S. Food and Drug Administration (FDA), fomivirsen(marketed as Vitravene) as a treatment for cytomegalovirus retinitis and mipomersen (marketed as Kynamro) for homozygousfamilial hypercholesterolemia.
Antisense RNA
Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribedwithin a cell. Some authors have used the term micRNA (mRNA-interfering complementary RNA) to refer to these RNAs but it is not widely used.
Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. This effect is therefore stoichiometric. An example of naturally occurring mRNA antisense mechanism is the hok/sok system of the E. coli R1 plasmid. Antisense RNA has long been thought of as a promising technique for disease therapy; the first antisense therapeutic to reach the market is the drug fomivirsen, approved in 1998. Mipomersen was approved in the united states in 2013. One commentator has characterized antisense RNA as one of “dozens of technologies that are gorgeous in concept, but exasperating in [commercialization]”. Generally, antisense RNA still lack effective design, biological activity, and efficient route of administration.
The effects of antisense RNA are related with the effects of RNA interference (RNAi). The RNAi process, found only in eukaryotes, is initiated by double-stranded RNA fragments, which may be created by the expression of an anti-sense RNA followed by the base-pairing of the anti-sense strand to the target transcript. Double-stranded RNA may be created by other mechanisms (including secondary RNA structure). The double stranded RNA is cleaved into small fragements by DICER, and then a single strand of the fragment is incorporated into the RNA-induced silencing complex (RISC) so that the RISC may bind to and degrade the complementary mRNA target. Some genetically engineered transgenic plants that express antisense RNA do activate the RNAi pathway. This processes resulted in differing magnitudes of gene silencing induced by the expression of antisense RNA. Well-known examples include the Flavr Savr tomato and two cultivars of ringspot-resistant papaya.
Transcription of longer cis-antisense transcripts is a common phenomenon in the mammalian transcriptome. Although the function of some cases have been described, such as the Zeb2/Sip1 antisense RNA, no general function has been elucidated. In the case of Zeb2/Sip1, the antisense noncoding RNA is opposite the 5’ splice site of an intron in the 5’UTR of the Zeb2 mRNA. Expression of the antisense ncRNA prevents splicing of an intron that contains a ribosome entry site necessary for efficient expression of the Zeb2 protein. Transcription of long antisense ncRNAs is often concordant with the associated protein-coding gene, but more detailed studies have revealed that the relative expression patterns of the mRNA and antisense ncRNA are complex.
Translation Blocking?
By sterically blocking the translation initiation complex, Morpholinos can knock down expression of many target sequences completely enough that after waiting for existing protein to degrade, the target protein band disappears from Western blots. Unlike many antisense types (e.g. siRNA, phosphorothioates), Morpholinos generally do not cause degradation of their RNA targets; instead, they block the biological activity of the target RNA until that RNA is degraded naturally, which releases the Morpholino. This means that RT-PCR is not suitable for assaying translation blocking by Morpholinos.
Splice Blocking?
By blocking sites involved in splicing pre-mRNA, Morpholinos can be used to modify and control normal splicing events. This activity can be conveniently assayed by RT-PCR, with successful splice-modification appearing as changes in the RT-PCR product band on an electrophoretic gel. The band might shift to a new mass or, if splice-modification triggers nonsense-mediated decay of the transcript, the band will lose intensity or disappear.
How to Delivery Morpholino?
Like all gene knockdown reagents, Morpholinos must be actively delivered into most cells. Morpholinos can be delivered into cultured cells by a variety of methods, including scrape-loading of adherent cells, electroporation, and even microinjection. However, our Endo-Porter delivery reagent generally gives excellent delivery in cultured cells in terms of the amount of Morpholino delivered per cell, even distribution throughout a population of cells, reproducibility of delivery and non-toxicity. For animal experiments our Vivo-Morpholinos with their attached delivery moiety enter into cells from the blood after i.v. administration or enter cells in tissues surrounding local injections; they are also the most convenient antisense oligos for use in cell cultures or explants, as they do not require any additional agent for cytosolic delivery.
The Structure od Morpholino.
Segment of a Morpholino-RNA heteroduplex, 8-mer shown:
A Morpholino oligo is radically different from natural nucleic acids, with morpholine rings replacing the ribose or deoxyribose sugar moieties and non-ionic phosphorodiamidate linkages replacing the anionic phosphates of DNA and RNA. Each morpholine ring suitably positions one of the standard DNA bases (A,C,G,T) for pairing, so that a 25-base Morpholino oligo strongly and specifically binds to its complementary 25-base target site in a strand of RNA via Watson-Crick pairing. Because the uncharged backbone of the Morpholino oligo is not recognized by enzymes or signaling proteins, it is completely stable to nucleases and does not trigger an innate immune response through the Toll-like receptors. This avoids oligo degradation, inflammation and interferon induction, which are problems commonly encountered with other gene knockdown reagents.
Some General Advice for Morpholino Experiments: Assessing delivery
As with all gene knockdown agents, effective delivery of Morpholinos is crucial. While confirming delivery is not crucial for experiments using embryonic microinjection, confirming delivery is important for experiments with cultured cells. Our Endo-Porter delivery reagent generally gives simple, reliable delivery for most cell types. You can assess whether you are getting good delivery into the cytosol of your cells by using a fluorescent-tagged Morpholino. We recommend you use our inexpensive fluorescein-tagged Standard Control oligo for this purpose.
When using a fluorescent Morpholino to confirm delivery, start with a concentration of 10 microMolar Morpholino oligo in the culture medium. This is a high enough concentration that after 16 hours the fluorescent-tagged Morpholino should be visible in the cytosol by fluorescence microscopy. An inverted fluorescent microscope is the best choice for observing cells in culture plates. Observe live (unfixed) cells. Use a dry objective lens with the highest available numerical aperture to gather as much light as possible from the cells. Successful delivery is manifested by diffuse fluorescence throughout the cytosol. Punctate fluorescence usually indicates that the oligo is trapped in endosomes, so ignore punctate spots and look for the diffuse fluorescence.
For actual experiments delivering your custom-sequence Morpholino with Endo-Porter you generally need a concentration of only 1 to 5 microMolar for effective steric blocking of most mRNAs.
Advice for Morpholino Experiments: Solubility and Morpholinos
Though Morpholino oligos are much more soluble than other non-ionic structural types (such as PNAs), some Morpholinos with high G content (>30%) do have limited solubility. Also, attachment of our red-emitting lissamine fluor sometimes decreases oligo solubility. Freeze-thaw cycles or long-term storage at 4C can cause slow precipitation or container association of Morpholinos and decrease the solution activity. This can sometimes be reversed by heating 5 min at 65C or by autoclaving (use the liquid cycle). We recommend that Morpholinos be stored at room temperature, which helps to keep the oligos in solution; be careful to avoid drying out the oligos. Making Morpholino stock solutions at or below 1 milliMolar helps to avoid solubility problems.
DNA, RNA, and most gene knockdown reagents are typically stored in an ice bath during experiments to minimize degradation by nucleases. Because Morpholinos are completely resistant to enzymatic degradation and aqueous solutions are chemically stable indefinitely at room temperature, we recommend that during your experiments you keep your stock solutions of Morpholinos at room temperature. Chilling your stock solutions in an ice bath simply risks causing the Morpholino to precipitate out.
If you are keeping Morpholinos as aqueous stock solutions and notice a decrease in antisense activity of the solution, you can autoclave the stock (use the liquid cycle of the autoclave). The oligos are very heat stable and can tolerate several rounds of autoclaving without degradation. Often autoclaving will revive a stock that has lost some activity due to aggregation of oligo.
For long-term storage your Morpholino stock solutions are best stored lyophilized, The next-best choice is storage in solution at room temperature. Wrapping the closure of the vial in Parafilm can help minimize microbial contamination and evaporation. Be careful to keep the oligo from drying out, as it can be difficult or impossible to redissolve a dried-down oligo (though freeze-dried oligos dissolve fairly easily). Keeping the vial in a dark box is a good practice, as fluor-labeled oligos can photobleach in light.
These precautions are more than needed for most sequences, but their routine use helps to avoid unpleasant surprises in the event you encounter a sequence with low solubility.
Why use a Vivo-Morpholino?
Vivo-Morpholinos* are the knockdown, exon-skipping or miRNA blocking reagent of choice for in vivo experiments. Outstanding results can be achieved systemically with intravenous (I.V.) injection, and modest systemic delivery achieved with Vivo-Morpholinos by intraperitoneal (I.P.) injection. Efficient localized delivery can be achieved by injecting the Vivo-Morpholino directly into the area of interest. Vivo-Morpholinos are also effective and easy to use in cultures.
Vivo-Morpholinos get the job done by combining two important parts:
Reference:
http://www.gene-tools.com/vivomorpholinos
A Vivo-Morpholino is comprised of a Morpholino oligo with a unique covalently linked delivery moiety, which is comprised of an octa-guanidine dendrimer. It uses the active component of arginine rich delivery peptides (the guanidinium group) with improved stability and reduced cost. The Vivo-Morpholino is assembled by coupling the vivo-delivery group to a Morpholino while the oligo is still bound to its synthesis resin, allowing excellent purification by washing the solid-phase resin. Vivo-Morpholinos must be chosen prior to synthesis and cannot be added later because the vivo-delivery group is added to a Morpholino prior to cleavage from its synthesis resin.
In vivo methods
For best systemic delivery results the injection method of choice is i.v. although i.p. can also be used. The maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period; dosing can be repeated daily, however, the sensitivity of organisms to the toxicity of the delivery moiety can vary depending on the type and health of the organism, so it is important to check the tolerance of your organism prior to dosing a group of animals. Local injections should be performed with reduced doses.
For short term experiments (3-day) Gene Tools suggests two days of i.v. injections at 12.5 mg/kg followed by analysis on day 3. Long term experiments should be optimized for your specific goals; however you can try starting with a loading dose equivalent to the short term experiment and then adjust dosage and times to find an optimum for continued delivery. Vivo-Morpholinos were optimized using 4 to 10 week old C57 Black mice and up to 20 mg/kg injections with successful outcomes. However, we have found that younger or older mice do not tolerate Vivo-Morpholinos as well and may require substantially lower doses. In addition the use of compromised mice, such as those with less robust genetic backgrounds, may require further dose limitations.
Cell culture methods
Vivo-Morpholinos are very simple to use in cultured cells. The typical concentration range for Vivo-Morpholinos in cell culture is 1 to 10 micromolar of Morpholino oligo in the culture medium. Concentrations higher than 10 micromolar could be toxic to cells. The protocol is as follows: add an appropriate amount of the solution to the culture medium on cells to reach the desired final concentration of Morpholino oligo, swirl to mix and harvest the cells as early as 24hr after treatment. It is not necessary to change the culture medium after adding oligos if the oligos are not toxic to the cells. If toxicity is observed, the oligo containing medium can be removed 2 to 4 hrs after treatment and fresh medium added. Serum significantly inhibits the efficacy of Vivo-Morpholinos so use a lower serum concentration if the cells tolerate it. The effect of splice-targeted oligos can be detected by reverse transcriptase PCR. The effect of translation blocking oligos can be assessed by analyzing protein levels by Western blot. The time needed to detect changes in the amount of protein translated from the targeted RNA will vary depending on the half-life of the protein, as preexisting protein must degrade for a knockdown to be evident.
open-access introduction to Vivo-Morpholinos:
http://www.gene-tools.com/vivomorpholinos
The secretin-secretin receptor axis modulates liver fibrosis through changes in TGF-β1 biliary secretion
Paul A. Morcos, Yongfu Li, and Shan Jiang. Vivo-Morpholinos: A non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. BioTechniques. 2008 Dec;45(6):616-26.
Kotb AM, Simon O, Blumenthal A, Vogelgesang S, Dombrowski F, Amann K, Zimmermann U, Endlich K, Endlich N. Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin. PLoS One. 2016 May 3;11(5):e0153768. doi: 10.1371/journal.pone.0153768. eCollection 2016.
“For in vivo KD, a volume of approximately 6 nl injection buffer was injected into the cardinal vein of 3dpf larvae.”
Costales MG, Rzuczek SG, Disney MD. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA. Bioorg Med Chem Lett. 2016 Apr 11. pii: S0960-894X(16)30386-9. doi: 10.1016/j.bmcl.2016.04.025. [Epub ahead of print]
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Nizzardo M, Simone C, Rizzo F, Ulzi G, Ramirez A, Rizzuti M, Bordoni A, Bucchia M, Gatti S, Bresolin N, Comi GP, Corti S. Morpholino-mediated SOD1 reduction ameliorates an amyotrophic lateral sclerosis disease phenotype. Sci Rep. 2016;6:21301. doi:10.1038/srep21301
Unmodified Morpholinos worked better than Vivo-Morpholinos in this mouse-brain application.
Schulman S, Bendapudi P, Sharda A, Chen V, Bellido-Martin L, Jasuja R, Furie BC, Flaumenhaft R, Furie B. Extracellular Thiol Isomerases and Their Role in Thrombus Formation. Antioxid Redox Signal. 2016 Jan 1;24(1):1-15. doi: 10.1089/ars.2015.6530. Epub 2015 Nov 18.
McMillin M, Frampton G, Quinn M, Ashfaq S, de los Santos III M, Grant S, DeMorrow S. Bile Acid Signaling Is Involved in the Neurological Decline in a Murine Model of Acute Liver Failure. Am J. Pathol. 2015;186(2):312–23. doi:10.1016/j.ajpath.2015.10.005
Vivo-Morpholinos delivered by intracerebroventricular infusion into mice.
Dewaele M, Tabaglio T, Willekens K, Bezzi M, Teo SX, Low DHP, Koh CM, Rambow F, Fiers M, Rogiers A, Radaelli E, Al-Haddawi M, Tan SY, Hermans E, Amant F, Yan H, Lakshmanan M, Koumar RC, Lim ST, Derheimer FA, Campbell RM, Bonday Z, Tergaonkar V, Shackleton M, Blattner C, Marine J-C, Guccione E. Antisense oligonucleotide-mediated MDM4 exon 6 skipping impairs tumor growth. J Clin Invest. 2015;[Epub ahead of print] doi:10.1172/JCI82534
Wong TT, Zohar Y. Production of reproductively sterile fish by a non-transgenic gene silencing technology. Sci Rep. 2015 Oct 29;5:15822. doi: 10.1038/srep15822.
Garg A, Wesolowski D, Alonso D, Deitsch KW, Ben Mamoun C, Altman S. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum. Proc Natl Acad Sci U S A. 2015 Sep 8. pii: 201515864. [Epub ahead of print]
Bednarek D, González-Rosa JM, Guzmán-Martínez G, Gutiérrez-Gutiérrez Ó, Aguado T, Sánchez-Ferrer C, Marques IJ, Galardi-Castilla M, de Diego I, Gómez MJ, Cortés A, Zapata A, Jiménez-Borreguero LJ, Mercader N, Flores I. Telomerase Is Essential for Zebrafish Heart Regeneration. Cell Rep. 2015 Sep 8;12(10):1691-703. doi: 10.1016/j.celrep.2015.07.064. Epub 2015 Aug 28.
Delivery into zebrafish hearts.
Grande MT, Sánchez-Laorden B, López-Blau C, De Frutos CA, Boutet A, Arévalo M, Rowe RG, Weiss SJ, López-Novoa JM, Nieto MA.Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat Med. 2015 Sep;21(9):989-97. doi: 10.1038/nm.3901. Epub 2015 Aug 3.
Salewsky B, Hildebrand G, Rothe S, Parplys AC, Radszewski J, Kieslich M, Wessendorf P, Krenzlin H, Borgmann K, Nussenzweig A, Sperling K, Digweed M. Directed Alternative Splicing in Nijmegen Breakage Syndrome: Proof of Principle Concerning Its Therapeutical Application. Mol Ther. 2015 Aug 12. doi: 10.1038/mt.2015.144. [Epub ahead of print]
Lam S, Chen H, Chen CK, Min N, Chu JJH. Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models. Sci Rep. 2015;5:12727 doi:10.1038/srep12727
Cator LJ, Pietri JE, Murdock CC, Ohm JR, Lewis EE, Read AF, Luckhart S, Thomas MB. Immune response and insulin signalling alter mosquito feeding behaviour to enhance malaria transmission potential. Sci Rep. 2015;5:11947 doi:10.1038/srep11947
Vivo-Morpholinos fed to mosquitos for gut knockdown
Carlson CG, Stein L, Dole E, Potter RM, Bayless D. Agents Which Inhibit NF-κB Signaling Block Spontaneous Contractile Activity and Negatively Influence Survival of Developing Myotubes. J Cell Physiol. 2015 Jun 30. doi: 10.1002/jcp.25085. [Epub ahead of print]
cell culture: murine primary myotubes
Xiang Y, Stine ZE, Xia J, Lu Y, O’Connor RS, Altman BJ, Hsieh AL, Gouw AM, Thomas AG, Gao P, Sun L, Song L, Yan B, Slusher BS, Zhuo J, Ooi LL, Lee CGL, Mancuso A, McCallion AS, Le A, Milone MC, Rayport S, Felsher DW, Dang CV. Targeted inhibition of tumor-specific glutaminase diminishes cell-autonomous tumorigenesis. J Clin Invest. 2015;[Epub ahead of print] doi:10.1172/JCI75836.
Ray D, Han Y, Franchitto A, DeMorrow S, Meng F, Venter J, McMillin M, Kennedy L, Francis H, Onori P, Mancinelli R, Gaudio E, Alpini G, Glaser SS. Gonadotropin-releasing hormone stimulates biliary proliferation by paracrine/autocrine mechanisms. Am J Pathol. 2015 Apr;185(4):1061-72. doi: 10.1016/j.ajpath.2014.12.004.
Kann M, Bae E, Lenz MO, Li L, Trannguyen B, Schumacher VA, Taglienti ME, Bordeianou L, Hartwig S, Rinschen MM, Schermer B, Benzing T, Fan CM, Kreidberg JA. WT1 targets Gas1 to maintain nephron progenitor cells by modulating FGF signals. Development. 2015 Apr 1;142(7):1254-66. doi: 10.1242/dev.119735.
Kumar V, Maurya VK, Joshi A, Meeran SM, Jha RK. Integrin Beta 8 (ITGB8) Regulates Embryo Implantation Potentially via Controlling the Activity of TGF-B1 in Mice. Biol Reprod. 2015 Mar 18. pii: biolreprod.114.122838. [Epub ahead of print] Vivo-Morpholino injected into uterine horn
Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schötterl S, Goedel A, Metzger K, Brade T, Parrotta E, Schaller M, Gerull B, Thierfelder L, Aartsma-Rus A, Labeit S, Atherton JJ, McGaughran J, Harvey RP, Sinnecker D, Mann M, Laugwitz K-L, Gawaz MP, Moretti A.Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy . EMBO Mol Med. 2015;[Epub ahead of print] doi:10.15252/emmm.201505047
Vivo-Morpholinos i.p. in mice
Koganti SR, Zhu Z, Subbotina E, Gao Z, Sierra A, Proenza M, Yang L, Alekseev A, Hodgson-Zingman D, Zingman L. Disruption of KATP channel expression in skeletal muscle by targeted oligonucleotide delivery promotes activity-linked thermogenesis. Mol Ther. 2015 Feb 4. doi: 10.1038/mt.2015.21. [Epub ahead of print]
Vivo-Morpholinos injected into leg muscle in mice
Echigoya Y, Aoki Y, Miskew B, Panesar D, Touznik A, Nagata T, Tanihata J, Nakamura A, Nagaraju K, Yokota T. Long-Term Efficacy of Systemic Multiexon Skipping Targeting Dystrophin Exons 45-55 With a Cocktail of Vivo-Morpholinos in Mdx52 Mice. Mol Ther Nucleic Acids. 2015 Feb 3;4:e225. doi: 10.1038/mtna.2014.76.
Ivanova AS, Shandarin IN, Ermakova GV, Minin AA, Tereshina MB, Zaraisky AG. The secreted factor Ag1 missing in higher vertebrates regulates fins regeneration in Danio rerio. Sci Rep. 2015 Jan 29;5:8123. doi: 10.1038/srep08123.
McClelland KS, Wainwright EN, Bowles J, Koopman P. Rapid screening of gene function by systemic delivery of morpholino oligonucleotides to live mouse embryos. PLoS One. 2015 Jan 28;10(1):e0114932. doi: 10.1371/journal.pone.0114932. eCollection 2015.
Rodrigues-Diez RR, Garcia-Redondo AB, Orejudo M, Rodrigues-Diez R, Briones AM, Bosch-Panadero E, Kery G, Pato J, Ortiz A, Salaices M, Egido J, Ruiz-Ortega M. The C-terminal module IV of connective tissue growth factor, through EGFR/Nox1 signaling, activates the NF-κB pathway and proinflammatory factors in vascular smooth muscle cells. Antioxid Redox Signal. 2015 Jan 1;22(1):29-47. doi: 10.1089/ars.2013.5500.
Hegde V, Hickerson RP, Nainamalai S, Campbell PA, Smith FJ, McLean WH, Pedrioli DM. In vivo gene silencing following non-invasive siRNA delivery into the skin using a novel topical formulation. J Control Release. 2014 Dec 28;196:355-62. doi: 10.1016/j.jconrel.2014.10.022. Epub 2014 Oct 30.
mouse intradermal paw injection
Heyland A, Hodin J, Bishop C. Manipulation of Developing Juvenile Structures in Purple Sea Urchins (Strongylocentrotus purpuratus) by Morpholino Injection into Late Stage Larvae. PLoS One. 2014 Dec 1;9(12):e113866. doi: 10.1371/journal.pone.0113866. eCollection 2014.
Stagg BC, Uehara H, Lambert N, Rai R, Gupta I, Radmall B, Bates T, Ambati BK. Morpholino-Mediated Isoform Modulation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) Reduces Colon Cancer Xenograft Growth. Cancers. 2014;6(4):2330-42. doi:10.3390/cancers6042330
mouse carrying human tumor xenograft
Prasad AA, McNally GP. Effects of Vivo Morpholino Knockdown of Lateral Hypothalamus Orexin/Hypocretin on Renewal of Alcohol Seeking. PLoS ONE. 2014;9(10):e110385. doi:10.1371/journal.pone.0110385
Microinfusion into lateral hypothalamus using a syringe pump
Berberoglu MA, Dong Z, Li G, Zheng J, Martinez LCGT, Peng J, Wagle M, Reichholf B, Petritsch C, Li H, Pleasure SJ, Guo S.Heterogeneously Expressed fezf2 Patterns Gradient Notch Activity in Balancing the Quiescence, Proliferation, and Differentiation of Adult Neural Stem Cells. J Neurosci. 2014;34(42):13911-23. doi: 10.1523/JNEUROSCI.1976-14.2014
zebrafish
Gallego-Villar L, Viecelli HM, Pérez B, Harding CO, Ugarte M, Thöny B, Desviat LR. A sensitive assay system to test antisense oligonucleotides for splice suppression therapy in the mouse liver. Mol Ther Nucleic Acids. 2014 Sep 16;3:e193. doi: 10.1038/mtna.2014.44.
Sundaramoorthi H, Khandekar G, Kim S, Jagadeeswaran P. Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish thrombocytes. Blood Cell Molec Dis. 2014;[Epub ahead of print] doi:10.1016/j.bcmd.2014.07.016
Francis H, McDaniel K, Han Y, Liu X, Kennedy L, Yang F, McCarra J, Zhou T, Glaser S, Venter J, Huang L, Levine P, Lai J-M, Liu C-G, Alpini G, Meng F. Regulation of the extrinsic apoptotic pathway by microRNA-21 in alcoholic liver injury. J Biol Chem. 2014;[Epub ahead of print] doi:10.1074/jbc.M114.602383
Sud R, Geller ET, Schellenberg GD. Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy For Tauopathies. Mol Ther Nucleic Acids. 2014 Jul 29;3:e180. doi: 10.1038/mtna.2014.30.
mice, i.m. injection
McMillin M, Galindo C, Pae HY, Frampton G, Di Patre PL, Quinn M, Whittington E, DeMorrow S. Gli1 Activation and Protection Against Hepatic Encephalopathy is Suppressed by Circulating Transforming Growth Factor β1 in Mice. J Hepatol. 2014;[Epub ahead of print] doi:10.1016/j.jhep.2014.07.015
mice, intra-cortical infusion
Motamedi FJ, Badro DA, Clarkson M, Rita Lecca M, Bradford ST, Buske FA, Saar K, Hübner N, Brändli AW, Schedl A. WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors . Nat Commun. 2014 Jul 17;5:4444. doi: 10.1038/ncomms5444.
organ culture: mouse kidney
Laranjeiro R, Whitmore D. Transcription factors involved in retinogenesis are co-opted by the circadian clock following photoreceptor differentiation. Development. 2014 Jul;141(13):2644-56. doi: 10.1242/dev.104380. Epub 2014 Jun 12. “Intraocular injections were performed in anaesthetised 5 dpf larvae with 0.4 nl/eye 0.5 mM neurod translation-blocking vivo-morpholino … 24-27 h before dissection of larval eyes.”
Yuan Y, Zhang J, Chang Q, Zeng J, Xin F, Wang J, Zhu Q, Wu J, Lu J, Guo W, Yan X, Jiang H, Zhou B, Li Q, Gao X, Yuan H, Yang S, Han D, Mao Z, Chen P, Lin X, Dai P. De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome. Cell Res. 2014 Jun 10. doi: 10.1038/cr.2014.77. [Epub ahead of print] No abstract available.
Oligos used were Vivo-Morpholinos (confirmed with author)
Nazmi A, Mukherjee S, Kundu K, Dutta K, Mahadevan A, Shankar SK, Basu A. TLR7 is a key regulator of innate immunity against Japanese Encephalitis Virus infection. Neurobiol Dis. 2014 Jun 5. pii: S0969-9961(14)00154-5. doi: 10.1016/j.nbd.2014.05.036. [Epub ahead of print]
Pietri JE, Cheung KW, Luckhart S. Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos. Insect Mol Biol. 2014 May 28. doi: 10.1111/imb.12103. [Epub ahead of print]
Ferguson DP, Dangott JJ, Timothy Lightfoot JT. Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity. BioTechniques. 2014;56(5):251-6.
Pfefferli C, Müller F, Jaźwińska A, Wicky C. Specific NuRD components are required for fin regeneration in zebrafish. BMC Biology. 2014;(12):30 doi:10.1186/1741-7007-12-30
Vivo-MO injected into clipped zebrafish fins.
Tan CW, Chan YF, Quah YW, Poh CL. Inhibition of enterovirus 71 infection by antisense octaguanidinium dendrimer-conjugated morpholino oligomers. Antiviral Res. 2014;[Epub ahead of print] doi:10.1016/j.antiviral.2014.04.004
Vivo-MO in cell cultures.
Renzi A, Mancinelli R, Onori P, Franchitto A, Alpini G, Glaser S, Gaudio E. Inhibition of the liver expression of arylalkylamine N-acetyltransferase increases the expression of angiogenic factors in cholangiocytes. Hepatobiliary Surg Nutr. 2014;3(1):4-10. doi:10.3978/j.issn.2304-3881.2014.01.02
Shaked I, Hanna DB, Gleißner C, Marsh B, Plants J, Tracy D, Anastos K, Cohen M, Golub ET, Karim R, Lazar J, Prasad V, Tien PC, Young MA, Landay AL, Kaplan RC, Ley K. Macrophage Inflammatory Markers Are Associated With Subclinical Carotid Artery Disease in Women With Human Immunodeficiency Virus or Hepatitis C Virus Infection. Arterioscler Thromb Vasc Biol. 2014 Mar 20. [Epub ahead of print]
Nizzardo N, Simone C, Salani S, Ruepp M-D, Rizzo F, Ruggieri M, Zanetta C, Brajkovic S, Moulton HM, Müehlemann O, Bresolin N, Comi GP, Corti S. Effect of Combined Systemic and Local Morpholino Treatment on the Spinal Muscular Atrophy Δ7 Mouse Model Phenotype. Clin Therap. 2014. 36(3):340-56. doi:10.1016/j.clinthera.2014.02.004
Carraro G, Shrestha A, Rostkovius J, Contreras A, Chao CM, El Agha E, Mackenzie B, Dilai S, Guidolin D, Taketo MM, Günther A, Kumar ME, Seeger W, De Langhe S, Barreto G, Bellusci S. miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development. Development. 2014 Feb 19. [Epub ahead of print]
Glaser S, Meng F, Han Y, Onori P, Chow BK, Francis H, Venter J, McDaniel K, Marzioni M, Invernizzi P, Ueno Y, Lai JM, Huang L, Standeford H, Alvaro D, Gaudio E, Franchitto A, Alpini G. Secretin Stimulates Biliary Cell Proliferation by Regulating Expression of MicroRNA 125b and MicroRNA let7a in Mice. Gastroenterology. 2014 Feb 25. pii: S0016-5085(14)00241-8. doi: 10.1053/j.gastro.2014.02.030. [Epub ahead of print]
Two tail-vein injections at 30 mg/kg targeting microRNAs mmu-miR-125b or mmu-miR-let7
Reissner KJ, Gipson CD, Tran PK, Knackstedt LA, Scofield MD, Kalivas PW. Glutamate transporter GLT-1 mediates N-acetylcysteine inhibition of cocaine reinstatement. Addic Biol. 2014;[Epub ahead of print] doi:10.1111/adb.12127
Rat nucleus accumbens
Lee TKW, Cheung VCH, Lu P, Lau EYT, Ma S, Tang KH, Tong M, Lo J, Ng IOL. Blockade of CD47 mediated CTSS-PAR2 signaling provides a therapeutic target for hepatocellular carcinoma. Hepatology. 2014;[Epub ahead of print] doi:10.1002/hep.27070
Human xenograft in mice, intratumoral injection
Ferguson DP, Dangott LJ, Schmitt EE, Vellers HL, Lightfoot JT. Differential Skeletal Muscle Proteome of High and Low Active mice. J Appl Physiol. 1985. 2014 Feb 6. [Epub ahead of print]
Karkampouna S, Kruithof BP, Kloen P, Obdeijn MC, van der Laan AM, Tanke HJ, Kemaladewi DU, Hoogaars WM, ‘t Hoen PA, Aartsma-Rus A, Clark IM, Ten Dijke P, Goumans MJ, Kruithof-de Julio M. Novel Ex Vivo Culture Method for the Study of Dupuytren’s Disease: Effects of TGFβ Type 1 Receptor Modulation by Antisense Oligonucleotides. Mol Ther Nucleic Acids. 2014 Jan 21;3:e142. doi: 10.1038/mtna.2013.69.
Human explant cultures
Reissner KJ, Brown RM, Spencer S, Tran PK, Thomas CA, Kalivas PW. Chronic administration of the methylxanthine propentofylline impairs reinstatement to cocaine by a GLT-1-dependent mechanism. Neuropsychopharmacology. 2014 Jan;39(2):499-506. doi: 10.1038/npp.2013.223. Epub 2013 Aug 29.
Velu CS, Chaubey A, Phelan JD, Horman SR, Wunderlich M, Guzman ML, Jegga AG, Zeleznik-Le NJ, Chen J, Mulloy JC, Cancelas JA, Jordan CT, Aronow BJ, Marcucci G, Bhat B, Gebelein B, Grimes HL. Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity. J Clin Invest. 2014 Jan 2;124(1):222-36. doi: 10.1172/JCI66005. Epub 2013 Dec 16.
Bollinger RJ, Bujak, JK, Madsen SS, Tipsmark CK. Vivo-Morpholino Induced Knock-Down of Gill Na,K-ATPase Impairs Seawater Acclimation in Rainbow Trout. Society for Integrative and Comparative Biology 2014. (poster) P2.136 rxb031@email.uark.edu
Franzosa JA, Bugel SM, Tal TL, La Du JK, Tilton SC, Waters KM, Tanguay RL. Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects. FASEB J. 2013 Dec;27(12):4866-76. doi: 10.1096/fj.12-225524. Epub 2013 Aug 23.
“morpholino oligonucleotide (MO) designed against both the guide strand and dicer cleavage site of Danio rerio (dre)-miR-19”
Ramachandran A, McGill MR, Xie Y, Ni HM, Ding WX, Jaeschke H. The receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice. Hepatology. 2013 Dec;58(6):2099-108. doi: 10.1002/hep.26547. Epub 2013 Oct 11.
Khandekar G, Jagadeeswaran P. Role of hepsin in factor VII activation in zebrafish. Blood Cells Mol Dis. 2013 Aug 15. doi:pii: S1079-9796(13)00194-0. 10.1016/j.bcmd.2013.07.014. [Epub ahead of print]
Bitar MS, Abdel-Halim SM, Al-Mulla F. Caveolin-1 upregulation in diabetic fibroblasts and wounded tissues: implication for understanding the underlying mechanisms of non-healing diabetic ulcers. Am J Physiol Endocrinol Metab. 2013 Aug 13. [Epub ahead of print]
Gao Z, Cooper TA. Reexpression of pyruvate kinase M2 in type 1 myofibers correlates with altered glucose metabolism in myotonic dystrophy. PNAS USA. 2013; [Epub ahead of print] doi:10.1073/pnas.1308806110
Voskoboynik A, Newman AM, Corey DM, Sahoo D, Pushkarev D, Neff NF, Passarelli B, Koh W, Ishizuka KJ, Palmeri KJ, Dimov IK, Keasar C, Fan HC, Mantalas GL, Sinha R, Penland L, Quake SR, Weissman IL. Identification of a colonial chordate histocompatibility gene. Science. 2013 Jul 26;341(6144):384-7. doi: 10.1126/science.1238036.
Knockdown in Botryllus schlosseri,a colonial urochordate
Spatazza J, Lee HH, Di Nardo AA, Tibaldi L, Joliot A, Hensch TK, Prochiantz A. Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity. Cell Rep. 2013 Jun 11. doi:pii: S2211-1247(13)00231-3. 10.1016/j.celrep.2013.05.014. [Epub ahead of print]
Kizil C, Iltzsche A, Kaslin J, Brand M. Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides. J Vis Exp. 2013;(75):e50415, doi:10.3791/50415
Video protocol for cerebroventricular microinjection in adult zebrafish.
Martin NT, Nakamura K, Davies R, Nahas SA, Brown C, Tunuguntla R, Gatti RA, Hu H. ATM-Dependent MiR-335 Targets CtIP and Modulates the DNA Damage Response. PLoS Genet. 2013;9(5):e1003505. doi:10.1371/journal.pgen.1003505
Lymphoblastoid cell lines
Jacobi CLJ, Rudigier LJ, Scholz H, Kirschner KM. Transcriptional regulation by the Wilms tumor protein, Wt1, suggests a role of the metalloproteinase Adamts16 in murine genitourinary development. J Biol Chem. 2013;[Epub ahead of print] doi:10.1074/jbc.M113.464644
Murine organotypic cultures: kidney and gonads
Ferguson DP, Schmitt EE, Lightfoot JT. Vivo-Morpholinos Induced Transient Knockdown of Physical Activity Related Proteins. PLoS ONE. 2013;8(4):e61472. doi:10.1371/journal.pone.0061472
Mouse; bradykinin enhancing brain delivery
Stec DE, Storm MV, Pruett BE, Gousset MU. Antihypertensive Actions of Moderate Hyperbilirubinemia: Role of Superoxide Inhibition. Am J Hypertens. 2013 Mar 12. [Epub ahead of print] doi: 10.1093/ajh/hpt038
Uehara H, Cho Y, Simonis J, Cahoon J, Archer B, Luo L, Das SK, Singh N, Ambati J, Ambati BK. Dual suppression of hemangiogenesis and lymphangiogenesis by splice-shifting morpholinos targeting vascular endothelial growth factor receptor 2 (KDR). FASEB J, 2013 Jan; 27(1):76–85. doi:10.1096/fj.12-213835 .
Kizil C, Kyritsis N, Dudczig S, Kroehne V, Freudenreich D, Kaslin J, Brand M. Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3. Dev Cell. 2012. 23(6):1230-7. doi: 10.1016/j.devcel.2012.10.014.
Kyritsis N, Kizil C, Zocher S, Kroehne V, Kaslin J, Freudenreich D, Iltzsche A, Brand M. Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science. 2012 Dec 7;338(6112):1353-6. doi: 10.1126/science.1228773.
Lin JF, Pan HC, Ma LP, Shen YQ, Schachner M. The Cell Neural Adhesion Molecule Contactin-2 (TAG-1) Is Beneficial for Functional Recovery after Spinal Cord Injury in Adult Zebrafish. PLoS ONE. 2012;7(12):e52376. doi:10.1371/journal.pone.0052376 Vivo-Morpholino in Gelfoam at spinal cord injury: “Contactin-2 anti-sense MO significantly reduced contactin-2 protein levels by approximately 82% compared with control MO 10 days after MO treatment being set to 100% … . Even 3 weeks after MO treatment, contactin-2 protein levels continued to remain decreased to a level of 49% (data not shown). It is noteworthy in this context that MO inhibition has been observed to last for more than 6 weeks.”
Hughes CE, Radhakrishnan UP, Lordkipanidzé M, Egginton S, Dijkstra JM, Jagadeeswaran P, Watson SP. G6f-Like Is an ITAM-Containing Collagen Receptor in Thrombocytes. PLoS ONE. 2012;7(12):e52622. doi:10.1371/journal.pone.0052622
Vivo-Morpholinos injected into both adult zebrafish (>3 month) and zebrafish embryos (3dpf).
Malerba A, Kang JK, McClorey G, Saleh AF, Popplewell L, Gait MJ, Wood MJA, Dickson G. Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy. Mol Ther Nucleic Acids. 2012;1:e62. doi:/10.1038/mtna.2012.54
Frampton G, Ueno Y, Quinn M, McMillin M, Pae HY, Galindo C, Leyva-Illades D, Demorrow S. The novel growth factor, progranulin, stimulates mouse cholangiocyte proliferation via Sirtuin1-mediated inactivation of FOXO1. Am J Physiol Gastrointest Liver Physiol. 2012 Oct 18. [Epub ahead of print]
Vivo-Morpholino injected into mice.
Renzi A, Demorrow S, Onori P, Carpino G, Mancinelli R, Meng F, Venter J, White M, Franchitto A, Francis H, Han Y, Ueno Y, Dusio G, Jensen KJ, Greene JJ, Glaser S, Gaudio E, Alpini G. Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes. Hepatology. 2012 Oct 18. doi: 10.1002/hep.26105. [Epub ahead of print]
Vivo-Morpholino injected into rats.
Csányi G, Yao M, Rodriguez AI, Al Ghouleh I, Sharifi-Sanjani M, Frazziano G, Xiaojun H, Kelley EE, Isenberg JS, Pagano PJ.Thrombospondin-1 Regulates Blood Flow via CD47 Receptor–Mediated Activation of NADPH Oxidase 1. Arterioscler Thromb Vasc Biol. 2012;[Epub ahead of print] doi:10.1161/ATVBAHA.112.300031
Luo YJ, Su YH. Opposing Nodal and BMP Signals Regulate Left–Right Asymmetry in the Sea Urchin Larva. PLoS Biol. 2012;10(10): e1001402. doi:10.1371/journal.pbio.1001402
Chen SX, Cherry A, Tari PK, Podgorski K, Kwong YKK, Haas K. The Transcription Factor MEF2 Directs Developmental Visually Driven Functional and Structural Metaplasticity. Cell. 2012;151(1):41–55 doi:10.1016/j.cell.2012.08.028
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
Vivo-Morpholino targeting miRNA in mouse embryos.
Xu H, Oliveira-Sales EB, McBride F, Liu B, Hewinson J, Toward M, Hendy EB, Graham D, Dominiczak AF, Giannotta M, Waki H, Ascione R, Paton JF, Kasparov S. Upregulation of junctional adhesion molecule-A is a putative prognostic marker of hypertension. Cardiovasc Res. 2012 Aug 22. [Epub ahead of print]
Tail vein injection in juvenile rats.
Chen Y, Londraville R, Brickner S, El-Shaar L, Fankhauser K, Dearth C, Fulton L, Sochacka A, Bhattarai S, Marrs JA, Liu Q.Protocadherin-17 function in zebrafish retinal development. Dev Neurobiol. 2012 Aug 28. doi: 10.1002/dneu.22053. [Epub ahead of print]
Injection into eyes of zebrafish embryos.
Pal D, Dasgupta S, Kundu R, Maitra S, Das G, Mukhopadhyay S, Ray S, Majumdar SS, Bhattacharya S. Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance. Nat Med. 2012 Aug;18(8):1279-85. doi: 10.1038/nm.2851. Epub 2012 Jul 29
Vivo-MO to FetA or Tlr4, 5- to 6-week-old female BALB/c mice, “Twenty-five nmoles of VMO was delivered to tail vein through injection in each mouse for 5 consecutive days, one on each day.”
Yokota T, Nakamura A, Nagata T, Saito T, Kobayashi M, Aoki Y, Echigoya Y, Partridge T, Hoffman EP, Takeda S. Extensive and Prolonged Restoration of Dystrophin Expression with Vivo-Morpholino-Mediated Multiple Exon Skipping in Dystrophic Dogs. Nucleic Acid Therapeutics. 2012. [Epub ahead of print] doi:10.1089/nat.2012.0368
Lai BS, Witola WH, El Bissati KE, Zhou Y, Mui E, Fomovska A, McLeod R. Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections. PNAS. 2012;[Epub ahead of print] doi:10.1073/pnas.1208775109
Aoki Y, Yokota T, Nagata T, Nakamura A, Tanihata J, Saito T, Duguez SMR, Nagaraju K, Hoffman EP, Partridge T, Takeda S. Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery. PNAS. 2012;[Epub ahead of print] doi:10.1073/pnas.1204638109
Cutler CP, Smith D. Vivo-morpholino knockdown of aquaporin 1 (AQP1) protein expression in American eel (Anguilla rostrata) gastrointestinal tract. The Bulletin, MDI Biological Laboratory. 2012;51
Notch EG, Chapline C, Flynn E, Lameyer T, Lowell A, Sato D, Shaw JR, Stanton BA. Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus. Comp Biochem Physiol A Mol Integr Physiol. 2012 May 2. [Epub ahead of print]
Nazmi A, Mukhopadhyay R, Dutta K, Basu A. STING Mediates Neuronal Innate Immune Response Following Japanese Encephalitis Virus Infection. Sci Rep. 2012;2:347. Epub 2012 Apr 2.
3–4 weeks old BALB/c mice, 5 mg/kg Vivo-Morpholino once per day for five days.
Parra MK, Gallagher TL, Amacher SL, Mohandas N, Conboy JG. Deep intron elements mediate nested splicing events at consecutive AG-dinucleotides to regulate alternative 3’ splice site choice in vertebrate 4.1 genes. Mol Cell Biol. 2012 Apr 2. [Epub ahead of print]
Vivo-Morpholinos injected in mouse tail veins at 15mg/kg on two consecutive days.
Sartor GC, Aston-Jones GS. A Septal-Hypothalamic Pathway Drives Orexin Neurons, Which Is Necessary for Conditioned Cocaine Preference. J Neurosci. 2012;32(13):4623-31. doi:10.1523/JNEUROSCI.4561-11.2012
Vivo-Morpholinos administered though cannulae into rat brains.
Owen LA, Uehara H, Cahoon J, Huang W, Simonis J, Ambati BK. Morpholino-Mediated Increase in Soluble Flt-1 Expression Results in Decreased Ocular and Tumor Neovascularization. PLoS ONE. 2012;7(3):e33576. doi:10.1371/journal.pone.0033576
Liu Y, Yu H, Deaton SK, Szaro BG. Heterogeneous Nuclear Ribonucleoprotein K, an RNA-Binding Protein, Is Required for Optic Axon Regeneration in Xenopus laevis. J Neurosci. 2012;32(10):3563-3574. doi:10.1523/JNEUROSCI.5197-11.2012
“In uninjured eyes, it efficiently knocked down hnRNP K expression in only the RGCs, without inducing either an axotomy response or axon degeneration.”
Jakobson M, Lintulahti A, Arumäe U. mRNA for N-Bak, a neuron-specific BH3-only splice isoform of Bak, escapes nonsense-mediated decay and is translationally repressed in the neurons. Cell Death Dis. 2012 Feb 2;3:e269. doi: 10.1038/cddis.2012.4.
Cultured mouse neurons
Nakamura K, Du L, Tunuguntla R, Fike F, Cavalieri S, Morio T, Mizutani S, Brusco A, Gatti RA. Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia. Hum Mutat. 2012 Jan;33(1):198-208. doi: 10.1002/humu.21632. Epub 2011 Nov 9.
Cultured primary cells
Notch EG, Shaw JR, Coutermarsh BA, Dzioba M, Stanton BA. Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation. PLoS ONE. 2011;6(12): e29462. doi:10.1371/journal.pone.0029462
Intraperitoneal (IP) injection, 14 µg/g SGK1 translation-blocking vivo-morpholino.
Taniguchi-Ikeda M, Kobayashi K, Kanagawa M, Yu CC, Mori K, Oda T, Kuga A, Kurahashi H, Akman HO, DiMauro S, Kaji R, Yokota T, Takeda S, Toda T. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature. 2011 Oct 5;478(7367):127-31. doi: 10.1038/nature10456.
Tail-vein and local injections in a mouse model of Fukuyama muscular dystrophy
Kizil C, Brand M. Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells. PLoS ONE. 2011;6(11):e27395. doi:10.1371/journal.pone.0027395
Injection of up to 500 nl into cerebroventricular fluid at Vivo-Morpholino concentrations up to 500 µM.
Osorio FG, Navarro CL, Cadiñanos J, López-Mejía IC, Quirós PM, Bartoli C, Rivera J, Tazi J, Guzmán G, Varela I, Depetris D, de Carlos F, Cobo J, Andrés V, Sandre-Giovannoli AD, Freije JM, Lévy N, López-Otín C. Splicing-Directed Therapy in a New Mouse Model of Human Accelerated Aging. Sci. Transl. Med. 2011;3:106ra107 doi:10.1126/scitranslmed.3002847
6 mg/Kg each of 2 sequences (or 12 mg/Kg control sequence) were administered to progeria model mice by tail vein injection twice per week for 12 weeks, starting at six weeks of age.
Reissner KJ, Sartor GC, Vazey EM, Dunn TE, Aston-Jones G, Kalivas PW. Use of vivo-morpholinos for control of protein expression in the adult rat brain. J Neurosci Meth. 2012 Jan 30;203(2):354-60. doi: 10.1016/j.jneumeth.2011.10.009. Epub 2011 Oct 17.
Intracranial injections in rat brains of oligos targeting three different RNA targets, some though cannula and some by direct injection.
NOTE: Kate Reissner informs us that the doses reported in this paper in nmol should have been reported in pmol; that is, the reported doses are 1000x too high.
Shi YB, Hasebe T, Fu L, Fujimoto K, Ishizuya-Oka A. The development of the adult intestinal stem cells: Insights from studies on thyroid hormone-dependent amphibian metamorphosis. Cell Biosci. 2011 Sep 6;1(1):30.
Injection into stage 53/54 Xenopus tadpoles, knockdown in gut.
Zammarchi F, de Stanchina E, Bournazou E, Supakorndej T, Martires K, Riedel E, Corben AD, Bromberg JF, Cartegni L.Antitumorigenic potential of STAT3 alternative splicing modulation. Proc Natl Acad Sci U S A. 2011 Oct 17. [Epub ahead of print]
Injections of Vivo-Morpholinos in a mouse tumor model either intratumorally at a final concentration of 0.12 mg/30 μl or intravenously at 15 mg/kg. Oligos were administered 2x week for either 1 or 3 weeks.
Nakamura K, Du L, Tunuguntla R, Fike F, Cavalieri S, Morio T, Mizutani S, Brusco A, Gatti RA. Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia. Hum. Mutat. 2011;[Epub ahead of print] doi:10.1002/humu.21632
Splice correction in lymphoblastoid cell line from patient.
Quinn M, Ueno Y, Pae HY, Huang L, Frampton G, Galindo C, Francis H, Horvat D, McMillin M, Demorrow S. Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat. Am J Physiol Gastrointest Liver Physiol. 2012 Jan 1;302(1):G182-93. doi: 10.1152/ajpgi.00205.2011. Epub 2011 Oct 6.
Intracerebroventricular injection of Vivo-Morpholinos in rat, daily 4ug/rat/day for 7 days.
Taniguchi-Ikeda M, Kobayashi K, Kanagawa M, Yu CC, Mori K, Oda T, Kuga A, Kurahashi H, Akman HO, DiMauro S, Kaji R, Yokota T, Takeda S, Toda T. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature. 2011;478:127-31 doi:10.1038/nature10456
FCMD model mice, human FCMD lymphoblasts and myotubes.
Vorlová S, Rocco G, LeFave CV, Jodelka FM, Hess K, Hastings ML, Henke H, Cartegni L. Induction of Antagonistic Soluble Decoy Receptor Tyrosine Kinases by Intronic PolyA Activation. Mol Cell. 2011;(43)6:927-39.
Cultured cells: HeLa, MDA-231 & HUVEC.
Nazmi A, Dutta K, Basu A. RIG-I Mediates Innate Immune Response in Mouse Neurons Following Japanese Encephalitis Virus Infection. PLoS ONE. 2011;6(6): e21761. doi:10.1371/journal.pone.0021761
Wu B, Benrashid E, Lu P, Cloer C, Zillmer A, Shaban M, Lu QL. Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development. PLoS ONE. 2011;6(5):e19906. doi:10.1371/journal.pone.0019906
Kowalik L, Hudspeth AJ. A search for factors specifying tonotopy implicates DNER in hair-cell development in the chick’s cochlea.Dev Biol. 2011 Jun 15;354(2):221-31. Epub 2011 Apr 8.
Vivo-Morpholinos locally injected in embryonic chick ears at E6.
Tsonis PA, Haynes T, Maki N, Nakamura K, Casco-Robles MM, Yamada S, Miura T, Chiba C, Del Rio-Tsonis K. Controlling gene loss of function in newts with emphasis on lens regeneration. Nature Protocols. 2011;6:593–599 doi:10.1038/nprot.2011.341 pdf
Widrick JJ, Jiang S, Choi SJ, Knuth ST, Morcos PA. An octaguanidine-morpholino oligo conjugate improves muscle function of mdx mice. Muscle & Nerve 2011 [Epub ahead of print]
Parra MK, Gee S, Mohandas N, Conboy JG. Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice. J Biol Chem. 2011 Feb 25;286(8):6033-9. Epub 2010 Dec 14.
Vivo-Morpholinos evaluated in mice at Lawrence Berkeley National Laboratory.
Nazmi A, Dutta K, Basu A. Antiviral and Neuroprotective Role of Octaguanidinium Dendrimer-Conjugated Morpholino Oligomers in Japanese Encephalitis. PLoS Negl Trop Dis 2010;4(11): e892. doi:10.1371/journal.pntd.0000892
Viral knockdowns in mice.
Carrillo M, Kim S, Rajpurohit SK, Kulkarni V, Jagadeeswaran P. Zebrafish von Willebrand factor. Blood Cells Mol Dis. 2010 Oct 28. [Epub ahead of print]
Vivo-Morpholinos in adult zebrafish.
Guo Y, Ma L, Cristofanilli M, Hart RP, Hao A, Schachner M. Transcription factor Sox11b is involved in spinal cord regeneration in adult zebrafish. Neuroscience. 2010 Oct 14. [Epub ahead of print]
Vivo-Morpholino on Gelfoam applied to zebrafish spinal cord transection at time of injury.
Kang JK, Malerba A, Popplewell L, Foster K, Dickson G. Antisense-induced Myostatin Exon Skipping Leads to Muscle Hypertrophy in Mice Following Octa guanidine Morpholino Oligomer Treatment. Mol Ther. 2010 Oct 5. [Epub ahead of print]
Matsuda H, Shi YB. An Essential and Evolutionarily Conserved Role of Protein Arginine Methyltransferase 1 for Adult Intestinal Stem Cells During Postembryonic Development. Stem Cells. 2010 Sep 16. [Epub ahead of print]
Intraperitoneal injection in Xenopus laevis.
Azoitei N, Pusapati GV, Kleger A, Möller P, Küfer R, Genze F, Wagner M, van Lint J, Carmeliet P, Adler G, Seufferlein T. Protein kinase D2 is a crucial regulator of tumour cell-endothelial cell communication in gastrointestinal tumours. Gut. 2010 Aug 23. [Epub ahead of print]
Vivo-Morpholinos in mice.
Vera T, Stec DE. Moderate Hyperbilirubinemia Improves Renal Hemodynamics in Angiotensin II Dependent Hypertension. Am J Physiol Regul Integr Comp Physiol. 2010 Oct;299(4):R1044-9. Epub 2010 Jul 28.
Vivo-Morpholinos in mice.
Kreidberg JA. WT1 and kidney progenitor cells. Organogenesis. 2010 Apr;6(2):61-70. Knockdown of WT1 in murine kidney explants; review offering perspective on experiments in Hartwig et al., Development 2010 (below).
Lecce L, Day M, Murphy CR. Abstract 15. ICAM-1 Is Involved in Uterine Receptivity in Rats. The Society for the Study of Reproduction 2010 Annual Meeting.
Maki N, Suetsugu-Maki R, Sano S, Nakamura K, Nishimura O, Tarui H, Del Rio-Tsonis K, Ohsumi K, Agata K, Tsonis PA. Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo. FASEB J. 2010 Sep;24(9):3462-7. Epub 2010 May 11.
This describes Vivo-Morpholino use in a newt.
Presentations using Vivo-Morpholinos from 2010 American Society of Gene & Cell Therapy meeting:
Systemic Delivery of Antisense Morpholino Corrects RNA Mis-Splicing and Reduces Myotonia in a Transgenic Mouse Model of Myotonic Dystrophy Type 1, Wheeler et al.
See abstract 14 on this linked page.
Multiple Exon-Skipping Using Cell-Penetrating Morpholinos for Dystrophic Dogs, Yokota et al.
See abstract 16 on this linked page.
Hartwig S, Ho J, Pandey P, Macisaac K, Taglienti M, Xiang M, Alterovitz G, Ramoni M, Fraenkel E, Kreidberg JA. Genomic characterization of Wilms’ tumor suppressor 1 targets in nephron progenitor cells during kidney development. Development. 2010 Apr;137(7):1189-203.
Knockdown of WT1 in murine kidney explants.
Chablais F, Jazwinska A. IGF signaling between blastema and wound epidermis is required for fin regeneration. Development. 2010 Mar;137(6):871-9.
Injection of 1.5 mM Vivo-Morpholino into regenerating zebrafish fins.
Kim S, Radhakrishnan UP, Rajpurohit SK, Kulkarni V, Jagadeeswaran P. Vivo-Morpholino knockdown of alphaIIb: A novel approach to inhibit thrombocyte function in adult zebrafish. Blood Cells Mol Dis. 2010 Mar 15;44(3):169-74. Epub 2010 Jan 4.
Knockdown of alphaIIb in thrombocytes by i.v. injection of Vivo-Morpholinos into adult zebrafish.
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