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how to knock out a gene?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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how to knock out a gene?

Postby steelcat » Thu Oct 16, 2008 10:03 am

how to knock out a gene? *_*
And how to knock out a gene from a adult? -- is difficult.
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Re: how to knock out a gene?

Postby jonmoulton » Thu Oct 16, 2008 4:51 pm

Knockout is a difficult procedure, involving homologous recombination. The cre-lox system is often used for this. Once you have done the procedure and the animals are born, you screen for the knockout and breed together animals that carry the knocked-out gene to establish a true-breeding line. This can take a year.

Knockdowns are an alternative that is more convenient. You put in an antisense oligo or double-stranded RNA targeting the gene you want to knock down. In adult animals, you need a systemic delivery system. One example of systemic antisense are Vivo-Morpholinos (http://www.gene-tools.com//vivomorpholinos). These are transient knockdowns, they will not last for the life of the organism but may last long enough to be experimentally useful -- and they are much easier.

I work for Gene Tools and Vivo-Morpholinos are our product.
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Postby biologist34 » Fri Oct 17, 2008 9:32 am

Could you explain more about vivo-morholinos how does it work? and in laboratories whats its aims?To knockdown sphesific genes or i understood wrong about it?
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Re: how to knock out a gene?

Postby jonmoulton » Fri Oct 17, 2008 4:38 pm

There are two parts to this: (1) how do the Morpholinos work and (2) how does the delivery group on the end of the oligo help the Vivo-Morpholino get from the extracellular space into the cytosol/nuclear compartment of cells.

For (1), Morpholinos bind to complementary sequences in RNA and get in the way of other processes, like pre-mRNA splicing, mRNA translation or miRNA maturation. For more on their mechanism and use, here is a review article:
Moulton JD, Yan YL. Using morpholinos to control gene expression. Curr Protoc Mol Biol. 2008 Jul;Chapter 26:Unit26.8.

For (2), first some history. Peptides were discovered that can enter cells from the extracellar space, such as the Anntenapedia peptide of Drosophila or the Tat peptide of HIV. When Dr. Hong Moulton's group at AVI BioPharma attached the Tat peptide to a Morpholino oligo, it helped the Morpholino to enter cells:
Moulton HM, Hase MC, Smith KM, Iversen PL. HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev. 2003 Feb;13(1):31-43.
Variations on the peptide were tried, leading to development of arginine-rich peptides incorporating non-natural amino acids that delivered Morpholinos much more efficiently than the Tat peptide:
Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL, Moulton HM. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res. 2007;35(15):5182-91. Epub 2007 Aug 1.
Realizing that the arginine was crucial to the peptide's delivery activity and that it was the head-group of arginine, a guanidinium group, that interacted with the membrane to effect delivery, Dr. Yongfu Li of Gene Tools devised a new wholly-synthetic compound, a dendrimer with eight tips bearing a guanidinium at each tip, that might offer efficient delivery of attached to a Morpholino oligo. After demonstrating successful delivery in cell cultures and in animals, this was named the Vivo-Porter moiety and offered commercially as a modification of Morpholinos:
Li YF, Morcos PA. Design and Synthesis of Dendritic Molecular Transporter that Achieves Efficient in Vivo Delivery of Morpholino Antisense Oligo. Bioconjug Chem. 2008 Jul;19(7):1464-70. Epub 2008 Jun 20.

Studies on the arginine-rich peptide-Morpholino conjugates have shown that the oligos enter cells by endocytosis and then escape from the endosome. In some way, the peptide moieties render the endosomal membrane permeable. This may be due to the combined electrostatic attraction and hydrogen-bonding of the guanidiniums to the phosphate groups of phospholipids.
Moulton HM, Moulton JD. Peptide-assisted delivery of steric-blocking antisense oligomers. Curr Opin Mol Ther. 2003 Apr;5(2):123-32.
We think that a similar mechanism assists delivery of the Vivo-Morpholinos into cells, that is, endocytosis followed by distortion and permeabilization of the endosomal membrane.

I agree that the primary use of Vivo-Morpholino will be targeted knockdown of specific genes in adult animals. Morpholinos are generally used to explore the function of genes by knocking them down. You can explore this database to see how they have been used: pubs.gene-tools.com
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Postby biologist34 » Tue Oct 21, 2008 9:07 am

thanks for explanation
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