Hi everyone.
I've spent the last couple of months trying to optimise transfection of primary HCAEC and HUVEC with a Qiagen CMV-GFP positive control plasmid (eventually want to use ARE-GFP reporter) with not much luck. I've tried several reagents (Attractene/Lipofectamine 2000/Nanojuice/Viromer Red & Yellow/FuGeneHD) and several protocols (forward/reverse/density/ratios/plasmid amount etc) with no success, and substantial toxicity in the case of LF2000. Ideally I need relatively high efficiency (>50%) with minimal toxicity so I can use these cells for time lapse shear stress assays.
In the last month or so I started using the EaHy926 HUVEC cell line just to see if I could get anything at all (weeks of looking for GFP that isn't there gets dull!), and just tried with FuGene HD and Lipofectamine 2000 as that was what I had available. Even with optimisation, so far I've got max 10% efficiency with FuGene and LF2000 was highly toxic and not effective.
We've managed to get Qiagen to send a fresh vial of the positive control plasmid as that the only thing that's been constant throughout this, but to be honest I'm not really sure where to go from here. I'm personally leaning towards going down the lentiviral route, but supervisors are really keen for me to try to get this working via standard transfection given we have the reporter plasmid 'ready to go'.
Has anyone had any experience transfecting EC with these plasmids and can offer any advice?
Also, I'm getting in a trial of LF3000, but does anyone actually have any experience of whether it is more effective/less toxic than 2000?
Thanks in advance for any advice :)
maybe the vector has entered the cells but the cells do not like CMV promoter so you can not see GFP. Also you could try electroporation before you switch to lenti. Good luck.
Dear Phoebe,
Endothelial cells are difficult to transfect, as Yuning suggests electroporation is a good option using pcDNA 6.2 emeraldGFP (Invitrogen), see link below:
http://www.lifetechnologies.com/content/dam/LifeTech/migration/en/filelibrary/cell-culture/neon-protocols.par.27321.file.dat/huvec-vein.pdf
Perhaps reverse transfection will help
- coat your wells with 0,2% collagen,
- add your pDNA+LF2000 mixture into the well
- thereafter add your endothelial cells into the same well.
- Wash and refresh your medium 24 hrs afterwards.
HUVEC's are quite large of size, fluorescence can be dim, tried longer exposure time of microscope already? And see any difference in time 24hrs, 48hrs, 72 hrs after transfection?
Good luck,
T.J.
Hi Phoebe,
I recommend Magnetofection for primary endothelial cells; This technology has been very successful to transfect all kind of endothelial including HUVEC HCAEC etc
see here http://www.ozbiosciences.com/transfection-dna/22-polymag-magnetofection-reagent.html
here are few examples of publications:
http://www.ncbi.nlm.nih.gov/pubmed/20592186
http://www.ncbi.nlm.nih.gov/pubmed/22792348
http://www.ncbi.nlm.nih.gov/pubmed/25391818
http://www.ncbi.nlm.nih.gov/pubmed/16720831
If you need more information let me know at [email protected]
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