China2005NoAnimation

Information about China2005NoAnimation

Published on October 23, 2007

Author: FunSchool

Source: authorstream.com

Content

Morpholinos:  Morpholinos for gene knockdown and splice modification Jon D. Moulton, Ph.D. October 2005 Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Making a Protein:  Making a Protein Two main steps, transcription and translation Transcription: DNA is copied making mRNA. Translation: mRNA directs which amino acids to put into a protein Transcription Translation Making a Protein:  Making a Protein In the nucleus: DNA is copied making mRNA and introns are removed (spliced) from mRNA. In the cytosol: mRNA directs which amino acids to put in a protein. Transcription Translation Splicing Making a Protein:  Making a Protein Making a Protein:  Making a Protein Making a Protein:  Making a Protein Making a Protein:  Making a Protein Making a Protein:  Making a Protein Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Antisense:  Antisense Representative Antisense Structures:  Representative Antisense Structures RNA RISC dependant Morpholino Steric blocking Phosphorothioate RNase-H dependant Antisense Mechanisms:  Antisense Mechanisms RNase-H RISC Steric dependant dependant blocking Summerton J. Morpholino Antisense Oligomers: The Case for an RNase-H Independent Structural Type. Biochimica et Biophysica Acta 1999 1489: 141-158 Morpholino-RNA Heteroduplex:  Morpholino-RNA Heteroduplex Watson-Crick bonds Bases positioned for strong binding Usually 25 base Morpholino oligos are used Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes What is Translation?:  What is Translation? In the cytosol, an initiation complex forms on an mRNA and moves to the start codon (AUG). The large ribosomal subunit arrives and forms a complete ribosome. The ribosome moves along the mRNA, putting together amino acids to form a protein. What is Translation?:  What is Translation? Blocking Translation:  Blocking Translation A steric-blocking oligo stops the initiation complex as it moves toward the start codon. Without reaching the start codon, the complete ribosome cannot form. Without the complete ribosome, the protein will not be made. Making a Protein (review):  Making a Protein (review) Blocking Translation:  Blocking Translation Assaying Translation Blocking:  Assaying Translation Blocking Stancheva I, Collins AL, Van den Veyver IB, Zoghbi H, Meehan RR. A mutant form of MeCP2 protein associated with human Rett syndrome cannot be displaced from methylated DNA by notch in Xenopus embryos. Mol Cell. 2003 Aug;12(2):425-35. Western blot 10 ng Morpholino oligo microinjected into egg Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Blocking Splicing:  Blocking Splicing When a Morpholino binds to boundary of an exon and an intron, the Morpholino can change pre-mRNA splicing. This results in a changed mRNA and a changed protein. Making a Protein (review):  Making a Protein (review) Blocking Splicing:  Blocking Splicing Assaying Splice Blocking:  Assaying Splice Blocking RT-PCR product from primers 1 & 3, control and Morpholino-treated lanes Draper BW, Morcos PA, Kimmel CB. Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: A quantifiable method for gene knockdown. Genesis. 2001 Jul;30(3):154-6. Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Embryo Applications - UROD:  Embryo Applications - UROD Zebrafish (Danio rerio) example: Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37) Knockdown of UROD makes fluorescent uroporphyrinogen accumulate Embryo Applications - VEGF:  Embryo Applications - VEGF Embryo Applications - GFP:  Embryo Applications - GFP Embryo Applications – oep:  Embryo Applications – oep Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Delivery into Cytosol:  Delivery into Cytosol In most tissues, bare Morpholino oligos enter cells poorly. There are several good methods for enhancing delivery into the cytosol: Method Application Endo-Porter Cell cultures Peptide conjugates Cultures & in vivo Microinjection Embryos Scrape-loading Cell culture Electroporation Cultures & embryos Upregulation Test System:  Upregulation Test System A mutant splice site from human b-globin is fused to a luciferase gene (luciferase can make cells glow). Blocking the mutant splice site with an oligo edits out a stop codon and brings luciferase in-frame. Splice-blocking causes luciferase activity (cells glow). This is an excellent system for measuring delivery. Ryzard Kole developed a system to measure splice-blocking activity in HeLa cultures. Schmajuk G, Sierakowska H, Kole R. Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J Biol Chem 1999 Jul 30;274(31):21783-9 Upregulation Test System:  Upregulation Test System Endo-Porter:  Endo-Porter The Endo-Porter peptide delivers Morpholinos, peptides, or other weakly-charged cargo to the cytosol of cultured cells through an endocytotic pathway. Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Animal Applications:  Animal Applications Applications being developed for Morpholino oligos in animals include: Cancer (adenocarcinoma, breast & prostate) Cardiovascular applications: Prevention of restenosis after angioplasty Coronary artery bypass graft Treatment of muscular dystrophy Treatment of bacterial diseases Treatment of viral diseases (next page) Antiviral Applications:  Antiviral Applications Morpholino oligos are in clinical trials for: Flaviviridae: Hepacivirus: Hepatitis C virus Flaviviridae: Flavivirus: West Nile virus Morpholino oligos are being/have been tested against: Orthomyxoviridae: Influenzavirus: Influenza A virus Coronaviridae: Coronavirus: SARS virus Flaviviridae: Flavivirus: Dengue virus Filoviridae: Filovirus: Ebola virus Caliciviridae: Vesivirus Summary:  Summary Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes Publication database:  Publication database An online database at the Gene Tools website lists over 1040 publications using Morpholinos. Web address: pubs.gene-tools.com Morpholino Papers in Top Journals:  Morpholino Papers in Top Journals 55 in Nature Publishing Group (including 17 in Nature) 8 in Science 46 in Cell Press (including 8 in Cell) 39 in Journal of Biological Chemistry 25 in PNAS USA Morpholino Papers in Developmental Biology Journals:  Morpholino Papers in Developmental Biology Journals 161 in Development 114 in Developmental Biology 34 in Mechanisms of Development 24 in Developmental Dynamics 30 in Genesis Many developmental biology papers are in other journals Acknowledgements:  Acknowledgements Thanks to: The staff of Gene Tools, LLC; Richard Meehan for his Western blot image; Bruce Draper for his RT-PCR gel image; Jim Summerton, inventor of Morpholinos. Web resources: www.gene-tools.com Gene Tools, LLC www.avibio.com AVI Biopharma, Inc www.zfin.org Zebrafish Information Network Gene Tools, LLC Philomath, Oregon, USA:  Gene Tools, LLC Philomath, Oregon, USA

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