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Problems with gene knockdown

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Problems with gene knockdown

Postby zodiac1505 » Tue Sep 08, 2009 11:31 am

We use RNAi and Lentiviral transgenesis in our lab to generate transgenic mice. We use luceferase assay to validate the shRNA sequences. The problem is the results don't seem to traslate well into in vitro systems. I analysed 8 sequences with the assay and made virus with them. The virus / plasmid has an antibiotic resistance gene which is expressed under the same promoter control ( mir30 motif). shRNAs were tested against both rat and mouse cDNAs in luciferase assay. The infected cells (in both rat and mouse cell lines) were selected on antibiotic. But when I look at the protein expression in the cells by Western blot, there seem to be no reduction. Of the sequences, at least 3 showed significant knockdown in Luciferase assay. Does anyone know any other way to select shRNAs with good knockdown efficiency? I am really stumped at the moment. Any suggestion would be really appreciated. Thanx.
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Re: Problems with gene knockdown

Postby jonmoulton » Mon Sep 14, 2009 7:45 pm

An alternative is using antisense to knock down the gene expression. I work for Gene Tools, a company making Morpholino antisense oligos for researchers. Morpholinos can be designed to block translation of mRNA or to modify splicing of pre-mRNA. They are commonly used in zebrafish embryology, where the off-target gene modulation of siRNA is teratogenic. With a delivery moiety added to the oligo, a Vivo-Morpholino can be injected into an adult mouse, move from the blood to tissues and into cells and knock down translation or modify splicing.

http://en.wikipedia.org/wiki/Morpholino
http://www.gene-tools.com/vivomorpholinos
http://www.mdpi.com/1420-3049/14/3/1304

In:
http://www.biotechniques.com/BiotechniquesJournal/
Paul A. Morcos, Yongfu Li, and Shan Jiang. Vivo-Morpholinos: A non-peptide transporter delivers Morpholinos into a wide array of mouse tissues. BioTechniques. 2008 Dec;45(6):616-26.

Good luck with your knockdowns, I hope you find an effective method for your system.

- Jon
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