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Is it Possible


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#1 Richard.21

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Posted 20 July 2009 - 05:04 PM

Hi.....

Some kind of doubts have haunted my mind........ i have cells (HepG2) transfected (Fugene delivery system) with RNAi against Dengue virus structures. I make my selection using G418.
I made a stock of these cells line, last years using DMSO 10% Bovine serum 20% and Dmem medium. Stored at liquid nitrogen.
Recently i decide to thaw my cells from liquid nitrogen and then, my cells can't attach in the flasks. I turned to this forum on section of "tissue and cell culture". The cells are growing but they are in suspension.
Last weekend i talked to a friend about this and she wondered herself if any RNAi wouldn't be interfering (in cross reaction) with some kind of molecules of these cells and undergoing them to not attach.
Does anyone here have similar experience???

Thank you very much

Reis ,V.P.

#2 Jon Moulton

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Posted 21 July 2009 - 06:21 AM

It is certainly possible that the RNAi is suppressing expression of genes which encode proteins involved in substrate adhesion.

Comparison of siRNA-induced off-target RNA and protein effects. Aleman LM, Doench J, Sharp PA. RNA. 2007 Mar;13(3):385-95. Epub 2007 Jan 19.

3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A. Nat Methods. 2006 Mar;3(3):199-204.

Small interfering RNA-induced transcriptional gene silencing in human cells. Morris KV, Chan SWL, Jacobson SE, Looney DJ. Science 2004 Aug 27;305(5688):1289-92. Epub 2004 Aug 5.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.

Small RNAs with Imperfect Match to Endogenous mRNA Repress Translation: Implications for off-target activity of small inhibitory RNA in mammalian cells. Saxena S, Jonsson ZO, Dutta A. J Biol Chem. 2003 Nov 7;278(45):44312-44319.
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com




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