Hi everyone
I am try to transfect primary human keratinocytes for downstream use in a luciferase reaction. Thus far I have been trialling X-tremeGENE HP in a 1:3 and 1:2 ratio on keratinocytes >80% confluence with the luciferase being performed after 24 hours. Whilst patterns are clearly evident and repeatable in the luciferase, my RLU are maximum of 10-20. Has anyone transfected primary keratinocytes before and if so do you have a suggested protocol?
Many thanks
Tara,
I would say there is such a thing called "the best". I have had experience with X-tremegene, Fugene, Lipofectine, and so on. They all worked. The most often I used is called "PEI", which is the cheapest.
The transfection confluency is important. I normally control it in the range of 40% to 60%. Another thing I would suggest is to use eGFP to optimise your assay. It is worth to do some dose and time course experiment.
Dear Tara,
primary human keratinocytes can be considered as hard-to-transfect cells. Moreover, even if the transfection rate is high, this does not really imply that protein production will be as high.
This is why we generally recommend using the magnetofection technology*** that was demonstrated to be of importance for transfecting primary and hard-to-transfect cells. For you specific application you can refer to the article by Zhang et al, Nat Genet (ref in the link below).
Moreover, the magnetic nanoparticles formulation presents a high compaction level of nucleic acids; in this way, not only the rate of transfection is higher than with "classic" methods, but also level of transgene expression is enhanced. In this way you should observe enhanced luciferase expression.
Would you like to try this method, you can contact me via ResearchGate or directly at: [email protected],
best regards,
Cedric
MAGNETOFECTION
*** 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/22983302
Tara:
Fugene HD (http://fugenehd.com) is by far the best transfection reagent I have used. easy to use, works well on moue primary renal proximal tubule cells. But, I have no experience working with keratinocytes.
Good luck,
Prasad
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