Vivo-Morpholinos enter cells of adult animals

Vivo-Morpholinos take Morpholinos to the next level

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. If you wish to initially validate the oligo in cultured cells you can use the same Vivo-Morpholino in cultures. However, unmodified Morpholinos, delivered with either electroporation or Endo-Porter, are more efficient in cell cultures than Vivo-Morpholinos.

Vivo-Morpholinos get the job done with two important ingredients

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, low toxicity 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.

Vivo-Morpholinos are offered in three convenient amounts

All Vivo-Morpholinos are provided in 400, 2000, and 10000 nmole quantities as a liquid 0.5 mM stock in phosphate buffered saline ready for injection. For syntheses greater than 10000 nmol please contact Customer Support. All Vivo-Morpholinos are filtered and sterilized. The amount of oligo you will need depends on the experiment and the method of injection. For a typical 20g mouse injected at 12.5 mg/kg I.V. the dose is approximately 25 nmole per injection. This means you will get approximately 16 injections from the 400 nanomole quantity, 80 injections from the 2000 nanomole quantity and 400 injections from the 10000 nanomole quantity

The methods are simple

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. This can be repeated daily. 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.

Delivery to most major organs is achieved

Following the short-term delivery experiment described above, one can expect quantifiable knockdown or exon-skipping in liver, small intestine, colon, muscle, lung, and stomach tissues with lesser but quantifiable delivery in the spleen, heart, skin and brain. For assessment of specific tissues within an organ one can achieve greater results with direct localized injections at the same concentrations described.

You don’t need much from the lab

You will need to provide your own sterile syringes and needles (27 to 32 gauge). Vivo-Morpholinos are ready for you to inject without any other preparation.

You can perform deliveries based on the Morpholino oligo component

The molecular weight of the Morpholino oligo component is 1810 Daltons smaller than the whole Vivo-Morpholino. Your product information sheet provides the molecular weight of your Vivo-Morpholino. The 0.5 mM stock of your Vivo-Morpholino is approximately 4 mg/mL Morpholino component.

You might want to compare Vivo-Morpholino dose with a bare oligo dose. The specific calculation for mg/ml concentration of your Vivo-Morpholino in terms of the oligo component (without delivery moiety) is:
mg/mL Morpholino = (Mol. Wt. Vivo-Morpholino – 1810 Da) x 0.0005

Vivo-Morpholinos are stable

Vivo-Morpholinos are provided sterile and can be stored at room temperature or put in your refrigerator. If your Vivo-Morpholino becomes contaminated, it can be autoclaved or filter sterilized. To autoclave, disable the autoclave's vacuum cycle to prevent loss of liquid. To filter sterilize, use a 0.2 micron polysulfone membrane; do not use other membranes as they have higher affinity for Vivo-Morpholinos and their use can result in significant loss of oligo concentration. To minimize decrease in oligo concentration due to association with the membrane, use the smallest available syringe filter (13 mm or smaller).

Vivo-Morpholinos can be ordered online

You can log into our online store and order Vivo-Morpholinos (to log in, see the box in the left column on this page). For more information on how to order Vivo-Morpholinos Contact Us.

^*Patent pending

References

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For an open-access introduction to Vivo-Morpholinos, see:

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.

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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 Jul 28. [Epub ahead of print]

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 May 11. [Epub ahead of print]
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.

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. 2009 Dec 31. [Epub ahead of print]
Knockdown of alphaIIb in thrombocytes by i.v. injection of Vivo-Morpholinos into adult zebrafish.

Pérez B, Rincón A, Jorge-Finnigan A, Richard E, Merinero B, Ugarte M, Desviat LR. Pseudoexon exclusion by antisense therapy in methylmalonic aciduria (MMAuria). Hum Mutat. 2009 Sep 2. [Epub ahead of print]
Report of Vivo-Morpholinos compared with unmodified Morpholinos and Endo-Porter when tested in cultured human fibroblasts.

Moulton JD, Jiang S. Gene Knockdowns in Adult Animals: PPMOs and Vivo-Morpholinos. Molecules. 2009; 14(3):1304-1323.

Wu B, Li YF, Morcos PA, Doran TJ, Lu PJ and Lu QL. Octa-guanidine Morpholino Restores Dystrophin Expression in Cardiac and Skeletal Muscles and Ameliorates Pathology in Dystrophic mdx Mice. Mol. Ther. 2009 May;17(5):864-71. Epub 2009 Mar 10.

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.

Li YF, Morcos PA. Design and Synthesis of Dendritic Molecular Transporter that Achieves Efficient in Vivo Delivery of Morpholino Antisense Oligo. Bioconjug Chem. 2008 Jul;19(7):1464-70. Epub 2008 Jun 20.