siRNA in organotypic cultures - siRNA-mediated gene silencing in vitro and in vivo (Aug/18/2008 )

Hey all.
Im new to this forum and would be very greatful if anyone will be able to help me.
I have established a hippocampal organotypic culture system in my lab and now wish to treat it with siRNA.
Does anyone know if the treatment is any different than with cell culture, and is any work was done with this type of culture.
Thanks!!!

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Could this help? PMID: 14645201

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Hi!
I'm also working with organotypic cultures of hippocampus (interface culture method) and we're planning to silence genes with iRNA. I'm looking for papers, protocols and general info about iRNA technique (haven't done this before) but found that very few people are trying it on organotypic cultures or even brain slices .
I'm thinking about using viral vectors, because my experience with electroporation (pEGFP-N3 plasmid) shows I can't achieve a sufficient transfection.
Are you planning to silence genes in the whole tissue, or just in some areas of your interest?

Well, some papers I found:

http://ep.physoc.org/cgi/content/full/90/1/61

http://www.sciencedirect.com/science?_ob=A...0f8687f#sec0001

Keep me updated of what you find about it, 'cos I'm also looking for info...

Cheers,
SaRa

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This might be useful for you hippocampal folk.

PMID: 18761372

J Neurosci Methods. 2008 Aug 12. [Epub ahead of print]
Microinjection into cultured hippocampal neurons: A straightforward approach for controlled cellular delivery of nucleic acids, peptides and antibodies.
Lappe-Siefke C, Maas C, Kneussel M.

Microinjection is a standard method for delivery of Morpholino antisense for gene knockdown in embryos. I am uncertain if this is the "organotypic" system you are using, but if you know a developmental biologist there is a good chance they already have the gear needed for the microinjections.

Best wishes,

- Jon

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Hello,
Thanks for the info, Jon.
Although I can't download the whole article through ScienceDirect (can you?), I'm afraid they are using a hippocampal primary cultures, quite different from ours. Organotypic cultures in the interface method consist on a brain slice (250 to 400 um thickness) cultured onto a porous membrane. The tissue gets nutrients from the liquid medium beneath the membrane while it is oxigenated on its surface (in contact with the incubator atmosphere).

The problem is how to deliver the desired substance (siRNA, in this case) into the tissue. I've tried to apply antibodies on the culture surface, for example, and later found that only the upper part of the culture had received it. I've tried to electroporate the culture after microinjecting some DNA, but transfection is rather poor. That's why I was interested in viral vectors, as they might infect the culture in a way similar to an in vivo system. But I still don't know if these vectors will extend all over the tissue, or just keep restricted to the zone of application/injection. Even I don't know which viral vector is the best: AVV or LVV? All the work done in my lab about silencing genes has been done with LVV, but only on primary neuronal cultures or cell lines, so I'm a bit lost...

Well, any suggestion will be welcome.
Thanks,

SaRa.

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While I can't offer assurance that this system will work in hippocampal neurons, here is a knockdown technique for an organotypic system similar to what you describe (but using kidney explant tissue).

http://www.ncbi.nlm.nih.gov/pubmed/18476819

Biotechniques. 2008 Apr;44(4):547-9.

The use of Endo-Porter to deliver morpholinos in kidney organ culture.
Nikopoulos GN, Adams TL, Adams D, Oxburgh L, Prudovsky I, Verdi JM.

Maine Medical Center Research Institute, Center for Molecular Medicine, Scarborough, Maine 04074, USA.

Cellular interactions in development of the kidney are used as a model of reciprocal inductive events between epithelium and mesenchyme. Time- and labor-intensive methods have been developed to study this phenomenon. For example, in mice, the targeted disruption of genes in vivo has been used to modify the genetic program directing kidney development. However, gene targeting is a resource-intensive approach and alternative strategies for gene and protein modification in the kidney need to be developed. Herein, we have developed an efficient system for the delivery of antisense morpholino to alter normal protein expression. We describe the use of Endo-Porter to effectively deliver morpholinos to all parts and regions of the kidney explant. Also, we definitively show via confocal microscopy and Western blot analysis that the use of Endo-Porter in delivering antisense morpholinos is robust throughout the entire kidney explant, providing efficient suppression of protein expression. This method saves time and cost when compared with targeted disruption and is an improvement upon previous kidney organ culture methods.

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Hello,
I'm back again!

Thanks Jon for your reply. I have been reading about morpholinos and the reference you gave was very interesting, but I don't know if it is comparable with our model and the purposes we have. Finally, we've decided to try with lentiviral vectors. So I'm reading about it, and found an interesting website about lentiviral vector which is useful for dummies like me: http://biology.kenyon.edu/slonc/gene-web/L...l/Lentivi2.html

and also a paper about lentiviral vectors' use in organotipic slices of cerebellum:
http://www.sciencedirect.com/science?_ob=A...d9722fb26554a49

I hope this can help someone.
Of course, any feedback is welcome!

Cheers,

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