Lipofection is going to work in primary fibroblasts, but the efficiency will still be lower than for other cell types. We've worked to make a primary fibroblast transfection reagent (https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/) and we've also made complex condensers to compact liposome-encapsulated nucleic acids, which boosts transfection efficiency substantially. Although the efficiency for siRNA transfection can be high (over 80%) the efficiency for plasmid delivery will be far lower, although still enough if you're going to use selection protocols to isolate successfully-transfected cells.

Hi Bastian are you trying to get a transient transfection or generate stables? (I am guessing transient as you are using primary cells).

Fibroblasts are pretty tough critters so you could probably try Calcium phosphate precipitation.

It is fairly old technique where you basically precipitate the DNA out of solution onto the cells (and in the "old days" this type of technique used to be used to transfer entire chromosomes i.e. CMGT). It can be fairly toxic for some cells but as it is cheap (you can buy the components separately rather than buying a kit) it may be worth a shot.

Also it is worth double-checking (by sequencing) that you gene of interest is in-frame, in the correct orientation with regards to the promoter and does not contain any undesired point mutations (i.e. your transfection is working but your gene is not!).

Alternatively (if you have access to one) electroporation generally works for even large plasmids - although once again this can be damaging to cells.

Hope this helps,

Gary

Hey Gary,

thanks for your answer.

Yes, I am trying to get a transient transfection (CRISPR/Cas9). I already tried an electroporation, but with no success at all. The transfection of a smaller plasmid was successful.

Did you ever use a Calcium phosphate precipitation for your transfection and may have a protocol for it?

Best regards

Hi I haven't done this for...well, a long while!

Here is a basic protocol though

http://www.bio-protocol.org/e86

You could also buy kits too i.e.

https://www.thermofisher.com/uk/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/calcium-phosphate-co-precipitation.html

Did you get any signal from any of the transfection methods that you have tried with the large plasmid? I would expect a minimum of 1-2% transfection with lipo or electro.

If you got no signal at all (and you have not already done so) I would strongly suggest sequencing the insert (especially if it was generated from a PCR product).

Gary

Hey Gary,

thanks for your answer.

Yes, I reached an efficiency of about 2-5 % with lipo and electroporation.

But to go on, I need an efficiency of at least 40-60 %.  

Maybe, the old techniques will be the best ;)

Best regards

Bastian

Cool - at least you know it is in-frame. Luck with your experiment and fingers crossed!

Gary

Hi Bastian

I can suggest also another technology, it is called Magnetofection and has been successful in many primary cells and also with large plasmid you can get more information here http://www.ozbiosciences.com/content/13-magnetofection-transfection

I will suggest the Polymag Neo reagent for you and I recommend to look at the citation database

let me know if I can be of any further help

Good Luck and best Regards,

Olivier

Hey Daniyar,

until now, I just used a Calcium phosphate precipitation in HEK293T-cells, to transfect plasmid DNA. It worked really well.

Let me know, if you need a protocol for it.

Best regards

Bastian

I never tried it in fibroblasts. Did you?

Lipofectamine LTX works reasonable good (about 40 % efficiency).

Primary fibrobalsts are well transfected with our Viromer technology (Viromer BLUE for siRNA, Viromer RED or YELLOW for plasmids)

https://viromer-transfection.com/fibroblast-transfection

Lipofection is going to work in primary fibroblasts, but the efficiency will still be lower than for other cell types. We've worked to make a primary fibroblast transfection reagent (https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/) and we've also made complex condensers to compact liposome-encapsulated nucleic acids, which boosts transfection efficiency substantially. Although the efficiency for siRNA transfection can be high (over 80%) the efficiency for plasmid delivery will be far lower, although still enough if you're going to use selection protocols to isolate successfully-transfected cells.

and what about transfecting mRNA instead of plasmid DNA?

https://www.researchgate.net/project/VIROMERR-an-innovative-technology-for-transfection/update/5b7be5763843b0024406dd07