Morpholino Oligos:Morpholino Oligos: Making Sense of Antisense?g

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

Cincinnati, Ohio

Developmental Biology 243, 209–214 (2002)

08.12.2008 Genie Genetique – Sebastian König and Sebastian Olényi

OverviewOverviewDo nreg lating gene e pression helps• Downregulating gene expression helps understanding their functions

• Most of the experiments are done to understandMost of the experiments are done to understand embryonic development

• Morpholinos are synthetic, usually 25 bases long, ith diff t t twith a different sugar-structure

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

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

StructuresStructures

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

PrinciplePrinciple

“DGI reliably achieves >90% knockdown of 

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

gene expression”

AdvantagesAdvantagesE l hi h ffi d ifi it• Equal or higher efficacy and specificity greatly superior to any other antisense t t l tstructural 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 possibleg g p• No need for RNase H to ensure their activity

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

DisadvantagesDisadvantages

• Completely artificial molecule, so no vectors possiblep

• Delivery usually only via microinjection or electroporationelectroporation

• Not always high effects, uncertain amount of downregulation

• Side-effects for high dosage oligo-• Side-effects for high dosage, oligo-dependent

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

ApplicationApplication25 Littl lf l t it (l th 4• 25mers, Little self-complementarity (less than 4 contiguous intrastrand base pairs)

• Less than 36% guanine contentLess than 36% guanine content• No runs of more than triplets of GGG• Concentrations between 1 and 12µMConcentrations between 1 and 12µM• 2-4 days effectiveness• Controls withCo t o s t

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

t ith ff tparts with same effect– Other over- and/or underexpression studies

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi

Thank you!

ReferencesReferenceshttp://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

08.12.2008Genie Genetique – Sebastian König and 

Sebastian Olényi