For sensitive fibroblasts lipofection will be the least error-prone approach (see https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/). Larger plasmids will have an even more difficult time entering cells, so using a complex condenser will help improve the overall efficiency. If you have good selection protocols then you should still be able to end up with a homogenous population of cells.

If you're worried about damaging the membrane, electroporation isn't the method of choice. 

Never use electroporation in that case. Use lipoagents better.

Dear Joshua,

Two important points:

• fibroblasts can be considered hard-to-transfect cells and it's quite true that lipofection and classic transfection reagents show some limits.
• 12 kb length is quite long for a plasmid; "classic" plasmids for transfection are around 5 kb, and generally up to 9-10kb.

This is why, I would highly recommend you to try Magnetofection method*** that uses a Magnetic field to concentrate complexes of magnetic nanoparticles/nucleic acids onto the cell surface.

Even if it is considered as a physical way of transfecting cells, the Magnetofection uses the classic pathways to transfect cells (endocytosis...); no hole is created during the transfection process and cellular integrity remains intact.

All the material are biodegradable and compatible with cell and organisms life.

you can find more details in some references using Magnetofection to transfect fibroblasts: 

Frolov A., (J Biol Chem. 2013) : synovial fibroblasts transduced by magnetofection, Ji JH., (J Oral Pathol Med. 2009) : mouth epidermal fibroblasts transfected with siRNA by magnetofection or Couchoux H., (J Physiol. 2007) : fibroblasts from foetal fibers.

I would be very glad in assisting you trying the Magnetofection technology, so please do not hesitate to contact me either via Researchgate or directly at [email protected]

good luck with your experiments,

have a nice day,

Cedric

*** The Magnetofection technology uses a magnetic field to attract and concentrate complexes of magnetic nanoparticles and nucleic acid onto the cell surface as demonstrated by Grześkowiak BF et al, Pharm Res. 2014 Jul 18. (http://www.ncbi.nlm.nih.gov/pubmed/25033763).

http://www.ncbi.nlm.nih.gov/pubmed/23814065

http://www.ncbi.nlm.nih.gov/pubmed/19453842

http://www.ncbi.nlm.nih.gov/pubmed/17317753

Lipofectamine is preferable because it is easier to optimize the transfection conditions to meet your needs. It is easier to adjust the reagent concentration and transfection incubation time to achieve your goals. The amount of vector available may limit the number of conditions tried. I think the fact that the reagent can be incubated for different time periods is an advantage over electroporation.

Hello Joshua,

In a lecture we have used the old fashioned transfection with phosphate buffer because we used a GFP vector and primary endothelial blood vessel cells from Aorta samples. The Prof said this is the cheapest way and works better than other transfection methods in this cell model.

The advantage for your experiment would be that the phosphate buffer creates precipitates when the plasmid containing solution is added by pipetting it into the buffer solution and mix by up and down pipetting and immediately transfer the whole finished mix to the well wher your cells are seeded in. The phosphate-plasmid complex create a fall out and rains down on the attached cells and they take it up themselfes by phagocytosis, so no damage of the cell membranes at all.

Protocolls for this phosphate-plasmid transfection you should find many (often too many) in the web. I can also search my course stuff if you would like to have my one, it has worked well when I remember correct.

Good luck

For sensitive fibroblasts lipofection will be the least error-prone approach (see https://altogen.com/product/fibroblast-transfection-reagent-fibroblast-cells/). Larger plasmids will have an even more difficult time entering cells, so using a complex condenser will help improve the overall efficiency. If you have good selection protocols then you should still be able to end up with a homogenous population of cells.