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E Presentation Morpholino Oligos
 

E Presentation Morpholino Oligos

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Morpholino Oligos are an interesting technique to silence Genes. Learn more about their usage and potential.

Morpholino Oligos are an interesting technique to silence Genes. Learn more about their usage and potential.

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    E Presentation Morpholino Oligos E Presentation Morpholino Oligos Presentation Transcript

    • Morpholino Oligos: Making Sense of Antisense? g Based on the review of Janet Heasman Division 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
    • Overview • Do nreg lating gene e pression helps Downregulating expression understanding their functions • Most of the experiments are done to understand embryonic development • Morpholinos are synthetic, usually 25 bases long, with a diff ith different sugar-structure t t t • Typical model organisms: zebrafish, African clawed frog, frog sea urchin, chick and mice. urchin mice Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Structures Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Principle “DGI reliably  achieves >90%  knockdown of  gene expression” Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Advantages • E Equal or hi h efficacy and specificity l higher ffi d ifi it greatly superior to any other antisense structural type t t lt • Morpholinos do not activate the complement cascade or the interferon system and are not degraded • Excellent solubility and stability • Working at high concentrations is p g g possible • No need for RNase H to ensure their activity Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Disadvantages • Completely artificial molecule, so no vectors possible p • Delivery usually only via microinjection or electroporation • Not always high effects, uncertain amount of downregulation • Side-effects for high dosage oligo- dosage, dependent Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Application • 25 25mers, Little self-complementarity (l Littl lf l t it (less th 4 than 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 Co t o s t – mRNA rescue experiments – Two different morpholinos directed to different gene parts with same effect t ith ff t – Other over- and/or underexpression studies Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi
    • Genie Genetique – Sebastian König and  08.12.2008 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 Genie Genetique – Sebastian König and  08.12.2008 Sebastian Olényi