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How much Morpholino to inject into a zebrafish egg or early zygote?

An injection volume of 500 pL is reasonable (Rosen et al. 2009, http://www.jove.com/video/1115/microinjection-of-zebrafish-embryos-to-an...). The basic rule for dosing of Morpholino injections is to use the least dose that gives you the phenotype of interest. As the dose increases, so does the potential for off-target RNA interactions and, with that, the potential of seeing a phenotype that has nothing to do with your target RNA.

Wednesday, December 31, 2014 - 08:21
Intestinal delivery to zebrafish from fish water

Progatzky F, Sangha NJ, Yoshida N, McBrien M, Cheung J, Shia A, Scott J, Marchesi JR, Lamb JR, Bugeon L, Dallman MJ. Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation. Nat Commun. 2014 Dec 23;5:5864. doi: 10.1038/ncomms6864.

http://www.nature.com/ncomms/2014/141223/ncomms6864/full/ncomms6864.html

This paper claims delivery of Morpholino to the intestinal epithelium of zebrafish by putting Morpholinos in the fish water around feeding time.

Friday, December 26, 2014 - 13:17
Rescues with wild-type or mutant mRNA

Here is an interesting strategy for nailing down the relationship between a phenotype and a particular mutation. Using a zebrafish pnpla6 morphant, co-injection of wild-type human pnpla6 mRNA rescued the morphant phenotype while mutant human pnpla6 mRNA failed to rescue.

Wednesday, December 10, 2014 - 13:54
Of Mice and Morpholinos, 2014

These are some papers using Morpholinos in mouse, published in 2014.

Vivo-Morpholino

WT1 controls antagonistic FGF and BMP-pSMAD pathways in early renal progenitors . Motamedi FJ, Badro DA, Clarkson M, Rita Lecca M, Bradford ST, Buske FA, Saar K, Hübner N, Brändli AW, Schedl A. Nat Commun. 2014 Jul 17;5:4444. doi: 10.1038/ncomms5444.
Vivo-Morpholino in kidney organ culture.

Tuesday, October 28, 2014 - 11:12
Some Morpholino Citations for Hematology: 2014

Representative Recent Morpholino Citations for Hematology: 2014
Last edit: 4 Dec 2014

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.
Nuzzo F, Bulato C, Nielsen BI, Lee K, Wielders SJ, Simioni P, Key NS, Castoldi E. Haemophilia. 2014 Dec 3. doi: 10.1111/hae.12554. [Epub ahead of print].

Tuesday, October 21, 2014 - 13:34
Citations for Morpholino work with Ebola or Marburg filovirus

Following are citations of Ebola or Marburg filovirus drug development using Morpholino antisense oligos.

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Enterlein S, Warfield KL, Swenson DL, Stein DA, Smith JL, Gamble CS, Kroeker AD, Iversen PL, Bavari S, Muhlberger E. VP35 Knockdown Inhibits Ebola Virus Amplification and Protects against Lethal Infection in Mice. Antimicrob Agents Chemother. 2006 Mar;50(3):984-93.

Friday, September 5, 2014 - 11:12
APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly

Preitner N, Quan J, Nowakowski DW, Hancock ML, Shi J, Tcherkezian J, Young-Pearse TL, Flanagan JG. APC Is an RNA-Binding Protein, and Its Interactome Provides a Link to Neural Development and Microtubule Assembly. Cell. 2014 Jul 17;158(2):368-82. doi: 10.1016/j.cell.2014.05.042.

This paper reports rat brain in utero injection then electroporation of Morpohlinos as well as mouse experiments with peptide nucleic acids.

Tuesday, July 22, 2014 - 09:50
Oligos targeted to trigger nonsense-mediated decay (NMD)

This paper provides some experimental support for targeting steric-blocking oligos to alter splicing and trigger nonsense-mediated decay. The paper compared uniform 2'-methoxyethyl phosphorothioate oligos with 2'-MOE phosphorothioate gapmers (steric blocking versus RNase-H competent, respectively).

Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides.
Ward AJ, Norrbom M, Chun S, Bennett CF, Rigo F.
Nucleic Acids Res. 2014 May 1;42(9):5871-9. doi: 10.1093/nar/gku184. Epub 2014 Mar 3.

Thursday, June 12, 2014 - 15:38
Intranasal administration in mice of peptide-conjugated Morpholinos

Peptide-conjugated Morpholinos were administered intranasally to mice to alter the immune response to subsequent intranasal influenza doses. "BALB/c mice were anesthetized with ketamine, followed by i.n. administration with 100 mg (approximately 5 mg/kg) PPMO (in 40 ml of PBS)." Repeat dosing at 24 hours, influenza inoculation at 48 hours.

Monday, June 2, 2014 - 12:09
Knockdowns in mosquito guts

Vivo-Morpholinos were fed to mosquitos. The oligos decreased the amount of MAPK protein in the midgut and the number of Plasmodium oocysts in the mudgut.

"The feeding protocol described in the present study provides several advantages over current methods for gene knockdown in insects. The method is easy to apply, requires no special skills for delivery, and is highly target specific. ... This protocol should therefore be considered as an efficient alternative in studies requiring target specific protein knockdown in mosquitoes and other arthropods."

Thursday, May 29, 2014 - 11:45

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