11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Morpholino Oligos: Making Sense of Antisense?

Based on the review of Janet HeasmanDivision of Developmental Biology, Children’s Hospital Medical Center,

Cincinnati, Ohio

Developmental Biology 243, 209–214 (2002)

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Overview

• Downregulating gene expression helps understanding their functions

• Most of the experiments are done to understand embryonic development

• Morpholinos are synthetic, usually 25 bases long, with a different sugar-structure

• Typical model organisms: zebrafish, African clawed frog, sea urchin, chick and mice.

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Structures

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Principle

“DGI reliably achieves >90% knockdown of gene expression”

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Advantages

• Equal or higher efficacy and specificity greatly superior to any other antisense structural type

• Morpholinos do not activate the complement cascade or the interferon system and are not degraded

• Excellent solubility and stability• Working at high concentrations is possible• No need for RNase H to ensure their activity

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Disadvantages

• Completely artificial molecule, so no vectors possible

• Delivery usually only via microinjection or electroporation

• Not always high effects, uncertain amount of downregulation

• Side-effects for high dosage, oligo-dependent

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Application

• 25mers, Little self-complementarity (less than 4 contiguous intrastrand base pairs)

• Less than 36% guanine content• No runs of more than triplets of GGG• Concentrations between 1 and 12μM• 2-4 days effectiveness• Controls with

– mRNA rescue experiments– Two different morpholinos directed to different gene parts

with same effect– Other over- and/or underexpression studies

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

11.04.2023 Genie Genetique – Sebastian König and Sebastian Olényi

Thank you!

Referenceshttp://www.sanger.ac.uk/Info/Press/gfx/zebrafish.jpghttp://www.columbia.edu/itc/cerc/danoff-burg/invasion_bio/inv_spp_summ/xenopuslaevis.jpghttp://www.alaskaunderseatours.com/photos/Red-Sea-Urchin.jpg

http://www.gene-tools.com/products_and_applications http://www.bentham.org/ctmc/openaccessarticles/ctmc7-7/0003R.pdf