Morpholino use in Mosquitoes is Promising

Antisense technologies have had limited application because oligodexoynucleotides in vivo have very limited stability. This stability problem due to rapid degradation of short DNA molecules limits the bioavailability of the molecules. Over the years there has been an evolution of oligo design with the intention of increasing bioavailability without compromising Watson-Crick base-pairing. Early modifications were initially focused on conventional oligodeoxynucleotides and included modifications of the phophodiester linkage by the addition of sulfur (phosphorothioate), modifications of the ribose at the 2’ position (2’-O-methy; 2’-O-methoxythyl; 2’-O,4’-C methylene bridge (Locked Nucleic Acids, LNA); 2’-O,4’-C ethylene bridge (ENA). Then more radical modifications in which bases were linked by a backbone of amino acids (N-(2-aminoethyl)-glycine) call peptide nucleic acids and finally, morpholinos.

Morpholinos (phosphorodiamidate morpholino oligomers) are composed of 25 nucleotides with bases connected to a morpholino ring instead of ribose and instead of a phosphodiester bond there is a phosphorodiamidate bond.

Basic structure of a morpholino oligonucleotide

Basic structure of a morpholino oligonucleotide

Morpholinos work by binding to target mRNAs and interfering with either processing or translocation.

Morpholinos have been used effectively and often in Zebrafish and Xenopus embryological studies by directly injecting them into eggs or early embryos.

There have been no reports of them being used in insects until now. Drosophilists never picked up on this technology because they had other ways to modulate gene expression in embryos. Much of the currently available insect genetic technologies have diffused from the Drosophila community and it is not surprising that morpholinos have not been used as a tool in other insects. But knocking down genes in insect embryos (or any other tissue) can be a challenge and there are few tools available to do this in insects other than Drosophila. dsRNA injection into the hemocoel of insects has been used for many studies but it is a blunt instrument and alternatives when this method is ineffective are not available

Petri et al (2014) give morpholinos a try in the midgut of adult Anopheles stephensi and get some encouraging results.

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

If you are interested in reading about a heroic effort to use this technology in chick embryos along with a nice review of the technology check out this paper in a 2014 special issue of Developmental Biology.

Anneliese Norris, Andrea Streit (2014) Morpholinos: Studying gene function in the chick, Methods, Volume 66, Issue 3, 1 April 2014, Pages 454–465 Developmental Biology


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