Morpholino Oligos
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Morpholino Oligos: Making Sense of Antisense? Based on the review of Janet Heasman Division of Developmental Biology, Children’s Hospital Medical Center, Cincinnati, Ohio
Developmental Biology 243, 209–214 (2002)
05/22/09
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.
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Structures
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Principle
05/22/09
“DGI reliably achieves >90% knockdown of gene 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 05/22/09
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, oligodependent 05/22/09
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 05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Thank you! References http://www.sanger.ac.uk/Info/Press/gfx/zebrafish.jpg http://www.columbia.edu/itc/cerc/danoff-burg/invasion_bio/inv_spp_summ/xenopuslaevis.jpg http://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
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Developmental Biology 243, 209–214 (2002)
05/22/09
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.
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Structures
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Principle
05/22/09
“DGI reliably achieves >90% knockdown of gene 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 05/22/09
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, oligodependent 05/22/09
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 05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
Thank you! References http://www.sanger.ac.uk/Info/Press/gfx/zebrafish.jpg http://www.columbia.edu/itc/cerc/danoff-burg/invasion_bio/inv_spp_summ/xenopuslaevis.jpg http://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
05/22/09
Genie Genetique – Sebastian König and Sebastian Olényi
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