To prepare for an assay where several plasmids need to be transfected together, I am trying to optimize transfection into equine dermal fibroblasts with a constitutive GFP plasmid. I've tried lipofectamine 2000, TransIT LT1 & Turbofect but only got really low transfection efficiency. There are no other equine cell lines available unfortunately, so I have to use this one. Does anyone have a proven fibroblast transfection protocol they could share with me?
You could try Lonza electroporation kit. Often, it works well, particulary for primary cells.
Dear Alice,
Dermal fibroblasts are considered hard-to-transfect cells and it's quite true that lipofection and classic transfection reagents show some limits.
That's 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.
This methods is really efficient for primary cells and hard-to-transfect cells ; the best example will be the transfection of primary neurons, primary endothelial cells, primary amniotic cells...
Moreover, there are some references using Magnetofection to transfect fibroblast (but not equine dermal fibroblasts, I'm sorry). For more information you can have a look at :
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. the links are the following ones :
http://www.ncbi.nlm.nih.gov/pubmed/23814065
http://www.ncbi.nlm.nih.gov/pubmed/19453842
http://www.ncbi.nlm.nih.gov/pubmed/17317753
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
Hi,
for plasmid transfection of my fibroblasts (MEF, HEF) I use Viromer RED/YELLOW (from Lipocalyx).
- Badges
- Science method