standard Have You Considered Morpholinos?

Many are making efforts to move genetic technologies into insect systems where they have not been used before  and becoming painfully aware that some technologies may not be applicable to all insects. For example, dsRNA may work well when systemically inject into the hemocoel of one insect but not another. In fact, you can observe differences in efficacy of dsRNA-based gene silencing in different life-stages of the same insect (larva vs adult, for example).

Morpholino structure.

Morpholino structure.

While dsRNA is popular, often effective and relatively easy to prepare, it is not the only approach to the transient disruption of gene expression and it may in some cases be worth thinking about alternatives.

Before there was dsRNA, there were anti-sense oligonucleotides.

 

Chick Embryo

Chick Embryo

Norris and Streit (2014) review one class of gene-expression-modulating antisense oligonucleotides – morpholinos – and their application to the study of gene function in the developing chick.  Morpholinos have also been used extensively in the study of zebra fish embryogeneisis.

Morpholinos are oligonucleotides that do not have phosphodiester linkages between nucleotides but phosphorodiamididic bonds instead. Furthermore bases are not attached to a ribose or deoxyribose but to a morpholino ring.

This unique chemistry results in oligos that are stable, nuclease-resistant, efficacious, with long-term activity, water-soluble, low toxicity and highly specific. Typically morpholinos are 25mers and they have specificities that are claimed to be better than RNAi and siRNA.

Morpholinos can be used to target a variety of molecules including miRNAs, mRNA localization elements, mRNA splicing signals and others.

Morpholino blocking translation. image by Jon D. Moulton of Gene Tools, LLC

Morpholino blocking translation.
image by Jon D. Moulton of Gene Tools, LLC

Morpholino blocking splicing. image  by Jon D. Moulton of Gene Tools, LLC

Morpholino blocking splicing.
image by Jon D. Moulton of Gene Tools, LLC

 

Of course, delivery of these molecules remains a challenge, but one similar to delivering dsRNA or siRNA.

To facilitate delivery “Vivo-Morpholinos” have been developed that consist of your morpholino covalently linked to an octa-guanidine dendrimer. Morpholinos can also be linked to proteins which may also facilitate uptake by target cells.

Morpholinos can also be synthesized so that their activity can be light sensitive. By applying the appropriate wavelength of light one can turn gene expression on or off.

Norris and Streit (2014) review briefly how one designs morpholinos and then concentrate on describing in vivo electroporation methods in chick embryos which they use to deliver morpholinos to developing chick embryos.

Nematostella vectensis photo credit: G. Thomsen; Stony Brook University

Nematostella vectensis
photo credit: G. Thomsen; Stony Brook University

Many have delivered morpholinos by direct microinjection and effectively disrupted gene expression in a variety of systems. For a recent paper see Layden et al. (2014) who use morpholinos to investigate early development of the sea anemone Nematostella vectensis.

Whether any of this could be useful to insect biologists remains to be seen although Pietri et al (2014) recently reported the use of morpholinos in mosquitoes with some success.

It is probably fair to say that this technology is under-explored in insect systems.

Gene Tools is the sole commercial manufacturer selling research quantities of morpholinos world-wide and their website has a wealth of background information about these interesting oligonucleotides.

 

Norris A, Streit A (2014) Morpholinos: Studying gene function in the chick. Methods 66: 454-465 DOI: 10.1016/j.ymeth.2013.10.009

 

Other References Cites:

Layden M, Martindale M (2014) Non-canonical Notch signaling represents an ancestral mechanism to regulate neural differentiation. Evodevo 5: 30 doi:10.1186/2041-9139-5-30

J. E. Pietri, K. W. Cheung and S. Luckhart (2014) Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos. Insect Molecular Biology Article first published online: 28 MAY 2014DOI: 10.1111/imb.12103


 

 

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3 Comments

  1. Here’s a partial citation list for Morpholinos in insects

    McMahon A, Reeves GT, Supatto W, Stathopoulos A. Mesoderm migration in Drosophila is a multi-step process requiring FGF signaling and integrin activity. Development. 2010 Jul;137(13):2167-75.

    Coate TM, Swanson TL, Copenhaver PF. Reverse signaling by glycosylphosphatidylinositol-linked manduca ephrin requires a SRC family kinase to restrict neuronal migration in vivo. J Neurosci. 2009 Mar 18;29(11):3404-18.

    Coate TM, Wirz JA, Copenhaver PF. Reverse Signaling via a Glycosyl-Phosphatidylinositol-Linked Ephrin Prevents Midline Crossing by Migratory Neurons during Embryonic Development in Manduca. J Neurosci. 2008 Apr 9;28(15):3846-60.

    Pilot F, Philippe JM, Lemmers C, Lecuit T. Spatial control of actin organization at adherens junctions by a synaptotagmin-like protein Btsz. Nature. 2006 Aug 3;442(7102):580-4. Epub 2006 Jul 9.

    Bucher G, Klingler M. Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function. Development. 2004 Apr;131(8):1729-40. Epub 2004 Mar 17.

  2. This new paper and the earlier Anopheles paper both used Vivo-Morpholinos, an oligo linked to a guanidinium dendrimer that helps the oligo enter cells. They fed the Vivo-Morpholinos in solution to the insects and looked for knockdown of peptide expression in their guts.

    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
    http://www.nature.com/srep/2015/150708/srep11947/full/srep11947.html

    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]
    http://onlinelibrary.wiley.com/doi/10.1111/imb.12103/abstract

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