Morpholinos. for gene knockdown and splice modification. Jon D. Moulton, Ph.D. October 2005. Summary. Making a Protein Antisense Structure Blocking Translation Blocking Splicing Embryos Delivery to Cytosol Therapeutic Applications Publication Notes. Summary. Making a Protein
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for gene knockdown and splice modification
Jon D. Moulton, Ph.D. October 2005
Two main steps, transcription and translation
Transcription: DNA is copied making mRNA.
Translation: mRNA directs which amino acids
to put into 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.
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
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.
10 ng Morpholino oligo microinjected into egg
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.
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.
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.
Zebrafish (Danio rerio) example:
Knockdown of uroporphyrinogen decarboxylase (EC 188.8.131.52)
Endo-Porter Cell cultures
Peptide conjugates Cultures & in vivo
Scrape-loading Cell culture
Electroporation Cultures & embryos
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
The Endo-Porter peptide delivers Morpholinos, peptides, or other weakly-charged cargo to the cytosol of cultured cells through an endocytotic pathway.
An online database at the Gene Tools
website lists over 1040 publications
(including 17 in Nature)
(including 8 in Cell)
Many developmental biology papers are
in other journals
www.gene-tools.com Gene Tools, LLC
www.avibio.comAVI Biopharma, Inc
www.zfin.orgZebrafish Information Network