RNAi of two different genes - (Jun/05/2008 )

Hello all,
I was wondering if anybody knows of a method that you can use that would knock down two genes at the same time?? I have a gene that I think is being compensated for by another gene and I was told it doesn't work to knock down multiple genes?
Tara

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Morpholino oligos are routinely used to knock down multiple genes.

Ny A, Koch M, Vandevelde W, Schneider M, Fischer C, Diez-Juan A, Neven E, Geudens I, Maity S, Moons L, Plaisance S, Lambrechts D, Carmeliet P, Dewerchin M. Role of VEGF-D and VEGFR-3 in developmental lymphangiogenesis, a chemicogenetic study in Xenopus tadpoles. Blood. 2008 May 12. [Epub ahead of print]
http://www.ncbi.nlm.nih.gov/pubmed/18474726

Ishibashi H, Matsumura N, Hanafusa H, Matsumoto K, Robertis EM, Kuroda H. Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos. Mech Dev. 2008 Jan-Feb;125(1-2):58-66. Epub 2007 Oct 12.
http://www.ncbi.nlm.nih.gov/pubmed/18036787

Cermenati S, Moleri S, Cimbro S, Corti P, Del Giacco L, Amodeo R, Dejana E, Koopman P, Cotelli F, Beltrame M. Sox18 and Sox7 play redundant roles in vascular development. Blood. 2008 Mar 1;111(5):2657-66. Epub 2007 Dec 19.
http://www.ncbi.nlm.nih.gov/pubmed/18094332

Nousch M, Reed V, Bryson-Richardson RJ, Currie PD, Preiss T. The eIF4G-homolog p97 can activate translation independent of caspase cleavage. RNA. 2007 Mar;13(3):374-84. Epub 2007 Jan 19
http://www.ncbi.nlm.nih.gov/pubmed/17237356

Jasuja R, Voss N, Ge G, Hoffman GG, Lyman-Gingerich J, Pelegri F, Greenspan DS. bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis. Mech Dev. 2006 Jul;123(7):548-58. Epub 2006 May 25.
http://www.ncbi.nlm.nih.gov/pubmed/16824737

Campbell WA, Yang H, Zetterberg H, Baulac S, Sears JA, Liu T, Wong ST, Zhong TP, Xia W. Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss. J Neurochem. 2006 Feb 8; [Epub ahead of print]
http://www.ncbi.nlm.nih.gov/pubmed/16464238

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Can you do morpholinos on cells in culture-I think they may be just for fish and frogs

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I have been working with cultured cells and using RNAi for two gene knockdown recently and have had some success. We do one transfection on the first day then do a follow up transfection 24 hours later. Of course this will vary depending on the timeline of which gene is activated when and how efficiently and rapidly you can knockdown your gene of interest.

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Morpholinos are the dominant gene knockdown technology in fish and frogs due to the sensitivity of these systems to teratogenesis from off-target gene modulation. Because Morpholinos are more specific than other knockdown technologies, injection of a negative control sequence can result (after development) in a healthy adult fish, unlike the early lethality or misshapen embryos that result from most knockdown reagents. However, Morpholinos have also been used extensively in cell culture, and offer the same specificity of knockdown in culture applications.

Morpholinos can be delivered into cells in culture by electroporation, by the Endo-Porter endosomal release agent, by scrape loading (for adherent cells) or by covalently conjugated delivery moieties, like cell-penetrating peptides or Vivo-Morpholinos (though these are more expensive and designed for adult animal use).

Here is a protocol from Gene Tools and Amaxa describing delivery to cultures by electroporation:
http://www.gene-tools.com/files/Amaxa_Morp...gos_2008-04.pdf

and a paper describing electroporation in cultures:
Jubin R. Optimizing electroporation conditions for intracellular delivery of morpholino antisense oligonucleotides directed against the hepatitis C virus internal ribosome entry site. Methods Mol Med. 2005;106:309-22.

For a list of citations using Endo-Porter for delivery of Morpholinos in culture, see the lower part of this page:
http://www.gene-tools.com/node/24

Scrape-loading into adherent cells was described by Partridge et al. and has been used in many subsequent publications:
Partridge M, Vincent A, Matthews P, Puma J, Stein D, Summerton J. A simple method for delivering morpholino antisense oligos into the cytoplasm of cells. Antisense Nucleic Acid Drug Dev. 1996 Fall;6(3):169-75.

For covalently-conjugated cell-penetrating peptides, see:
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 Aug 1; [Epub ahead of print]

The first paper describing Vivo-Morpholinos has been accepted by Bioconjugate Chemistry and will be available as an Epub shortly.

You can access many more abstracts describing cell culture Morpholino experiments by searching on the keyword "culture" at pubs.gene-tools.com.

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