Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.
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Transcript of Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.
![Page 1: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/1.jpg)
Morpholinosfor gene knockdown and splice modification
Jon D. Moulton Summer 2008
![Page 2: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/2.jpg)
Gene Tools, LLCPhilomath, Oregon, USA
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Gene Tools LLCGene Tools makes Morpholino antisense oligos in Philomath, Oregon.
Researchers use Morpholinos to learn about the functions of proteins in cells.
We target a custom-made Morpholino to a particular mRNA to stop a cell from making a particular protein.
![Page 4: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/4.jpg)
Cells make proteins
• We’ll look at parts of a cell that are important for making proteins, then look at some of the steps that occur in a cell as it makes a protein.
• Morpholinos alter some of these steps. We’ll come back to Morpholinos soon.
![Page 5: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/5.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 6: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/6.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 7: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/7.jpg)
Some important biomolecules
• Proteins
• Nucleic acids
• Lipids
• Carbohydrates
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Some important biomolecules
• Proteins
structure, signaling, catalysis
• Nucleic acids
information, plans for proteins
• Lipids
separating compartments
• Carbohydrates
energy, signaling
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Plasma membrane
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Plasma membrane
The cell’s membrane is made of a two-molecule-thick
layer of detergent-like substance. Think of the membrane as a very stable soap bubble that separates
stuff inside from stuff outside.
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Plasma membrane
This space, inside the plasma membrane, is called the cytosol.
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Nuclear envelope
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Nuclear envelope
The nuclear envelope
and its contents are
called the nucleus.
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DNA
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DNA
DNA stores information needed for making proteins. The order of the DNA bases determines the order of amino acids in a protein.
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RNA
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RNA
An RNA molecule carries a temporary copy of the instructions for making a protein, shuttling the instructions from the nucleus to the cytosol.
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RNA
RNA is processed in the nucleus. Some parts, called introns, are spliced out. Other parts, exons, are sent to the cytosol.
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Ribosomes
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Ribosomes Ribosomes are the structures where the cell manufactures proteins.
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Protein
![Page 22: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/22.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 23: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/23.jpg)
Now we zoom in…
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Making a Protein
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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
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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
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Making a Protein
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Making a Protein (where the steps happen)
In the nucleus: TranscriptionDNA is copied into mRNA and introns are removed (spliced) from mRNA.
In the cytosol: TranslationOn a ribosome, mRNA directs which amino acids to put in a protein.
Transcription Translation
Splicing
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Making a Protein
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Making a Protein
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Making a Protein
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Making a Protein
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Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 34: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/34.jpg)
Antisense
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Antisense
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Antisense
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Morpholinobound to
RNA • Watson-Crick bonds
(A to T, C to G)
• Bases positioned for • strong binding
• Usually 25 base • Morpholino oligos• are used
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Morpholino bound to RNA
with the structure of the bases
shown
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Representative Antisense Structures
RNARISC
dependant
MorpholinoSteric
blocking
Phosphorothioate RNase-H dependant
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Antisense Mechanisms RNase-H RISC Steric
dependant dependant blocking
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Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 42: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/42.jpg)
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.
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What is Translation?
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What is Translation?
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What is Translation?
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What is Translation?
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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.
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Making a Protein (review)
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Blocking Translation
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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 blot10 ng Morpholino oligo microinjected into frog egg
![Page 51: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/51.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 52: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/52.jpg)
What is splicing?
![Page 53: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/53.jpg)
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 possibly a changed protein.
![Page 54: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/54.jpg)
Making a Protein (review)
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Blocking Splicing
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Assaying Splice Blocking
Lane from control fish is labeled 0, lane from fish treated with Morpholino is labeled 5
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.
DNA made by RT-PCR using primers 1 & 3, DNA size measured using gel electrophoresis
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Odd splices can happen
![Page 58: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/58.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 59: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/59.jpg)
Embryo Applications - URODZebrafish (Danio rerio) example:
Knockdown of uroporphyrinogen decarboxylase (EC 4.1.1.37)
Knockdown of URODmakes fluorescent uroporphyrinogen accumulate
HNNH
HNNH
O
OO
OO
O
O
OO
O O
O
O
O
O
O
URODHNNH
HNNH
O
O
O
OO
O
O
O
uroporphyrinogen III coproporphyrinogen III
-4 CO2
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Embryo Applications - VEGF
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Embryo Applications - GFP
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Embryo Applications – oep
![Page 63: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/63.jpg)
Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 64: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/64.jpg)
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 Vivo-Morpholinos Cultures & in vivoPeptide conjugates Cultures & in vivo
Microinjection EmbryosScrape-loading Cell culture
Electroporation Cultures & embryos
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Endo-Porter
The Endo-Porter peptide delivers Morpholinos, peptides, or other weakly-charged cargo to the cytosol of cultured cells through an endocytotic pathway.
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Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 67: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/67.jpg)
Animal Applications
Applications being developed in animals for Morpholino oligos include:
• Treatment of Duchenne muscular dystrophy (currently in clinical trials)
• Prevention of restenosis after angioplasty• Cancer (adenocarcinoma, breast & prostate)• Treatment of bacterial diseases• Treatment of viral diseases (next page)
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Antiviral Applications
Morpholino oligos are being/have been tested against:•Flaviviridae: Hepacivirus: Hepatitis C virus
•Flaviviridae: Flavivirus: West Nile virus•Orthomyxoviridae: Influenzavirus: Influenza A virus•Coronaviridae: Coronavirus: SARS virus •Flaviviridae: Flavivirus: Dengue virus •Filoviridae: Filovirus: Ebola virus
•Filoviridae: Filovirus: Marburg virus•Caliciviridae: Vesivirus
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Summary
Parts of animal cellsMaking a ProteinAntisense StructureBlocking TranslationModifying SplicingEmbryosDelivery to CytosolTherapeutic ApplicationsPublication Notes
![Page 70: Morpholinos for gene knockdown and splice modification Jon D. Moulton Summer 2008.](https://reader036.fdocuments.in/reader036/viewer/2022062517/56649e745503460f94b7478f/html5/thumbnails/70.jpg)
Resources on the web
Introduction:Morpholino article on Wikipedia http://en.wikipedia.org/wiki/Morpholino
More technical sites: www.gene-tools.com Gene Tools, LLC
www.avibio.com AVI Biopharma, Inc
www.zfin.org Zebrafish Information Network
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Publication database
An online database at the Gene Tools
website lists over 2300 publications
using Morpholinos (as of fall 2008).
Web address:
pubs.gene-tools.com
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AcknowledgementsThanks to:• The staff of Gene Tools, LLC for critique;• Richard Meehan for his Western blot image;• Bruce Draper for his RT-PCR gel image.
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Gene Tools, LLCPhilomath, Oregon, USA