Transfecting Primary Cells

We welcome any comments on you experiences with transfecting primary cell lines with cationic lipids and/or electroporation.

Here're are interesting comments of transfecting primary cells from Dr. Mark Behlke, CSO of IDT:

"Just wanted to give you a project update. We have good transfection conditions worked out for LTK cells and CHO cells now. Different reagents and different conditions proved to be optimal for each cell line.

The LTK cells proved to be a real nightmare. We were testing out different siRNAs and had a weird problem that it seemed that none of the siRNA worked even a little, with the weird observation that our controls were “very low”. After quite a bit of investigation, we finally figured out that the LTK cells were triggering an IFN pathway response to the siRNAs. Unfortunately, it was a bit odd and it took some time to figure out what was going on. The cells did not just “die” or show changes in the usually time frame expected for Type-I IFN responses, but clearly were activating new cytokine related transcription units. The promoter driving your transgene is regulated by the transcription factors induced, such that NTS1 mRNA levels increased upon transfection. The controls were “low”, showing basal levels, and the transfected cells were all “high”. It turns out that our dye-labeled transfection control sequence was triggering the pathway in a sequence-specific fashion. We are now leaving out the transfection control and things are OK. We can also totally evade the response using 2’OMe modified duplexes (we usually use these modified duplexes for siRNA intended for IV administration, since exposure to PBMCs usually triggers any/all possible IFN responses).

We can avoid these responses and now have the assay system working. So, we can finally start proper testing!"

Preliminary Results from Dr. Helen Hellmich of UTMB:

"It looks like the I-fect delivered the control labeled IDT oligos to approximately the right place in the rat hippocampus 48 hours post-injection. After my research assistant comes back from her vacation, we will try a longer time point, 72 hours and alter our stereotactic coordinates slightly to more directly target the CA3 subregion. After that we will try the real siRNA injections with I-fect, hopefully in the next few weeks. I will keep you all posted to whether this really works."

Protocols for Delivering siRNA to Neurons

Here's a link to the protocol: Transfecting Schwann Cells with i-Fect and below are related data.

Figure:si RNA-mediated suppression of target gene expression in Schwann cells. A, Detergent extracts of siNeg- or siGly1- transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top);aliquots of undigested extracts wereimmunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B, Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican- 1 antibodies (green) and DAPI (4',6'-diamidino-2 phenylindole) to stain nuclei (red). C, Schwann cells were transfected with siNeg or si 4(V)and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right);aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti- 4(V) collagen (top) or anti- -actin (b ottom) antibodies.

intrathecal in vivo delivery of siRNA(pdf - 57Kb)

Publications:

Progress Towards in vivo Use of siRNAs
siRNAi: Applications in Functional Genomics and Potential as Therapeutics
Assembly and Function of RNAi Silencing Complexes
Synthetic dsRNA Substrates Enhance RNAi Potency and Efficacy