Lipofection is not very effective in transfecting HUVEC that kind of slowing growing primary line. You may consider using lentiviral expression system. I actually tried infecting a few genes into HUVEC, it worked quite well. See if anyone at your institution you can ask for the materials and the technical support (culture hood for virus). That's not hard and it is not expensive, all you need is the expression, packaging, envelope vectors (available in Addgene) and the packaging 293T cells and any lipofection reagent for transfecting all these vectors into the 293Tcells.
If someone at your institute has an Amaxa Nucleofector that would do the job.
Otherwise check this article:
http://www.nature.com/protocolexchange/protocols/2096
Dear Michael,
I have already tried to transfect endothelial cells from brain capillary. The best way was Lipofectamine 2000 and of course the electroporation using nucleofector from lonza.
Let me know if you want more details
I will not say one method is better than others but my recent transfection using Lipofectamine LTX and Lipofectamine RNAiMAX was kind of working for my pulmonary artery endothelial cells. Some of my colleague also start to use them for bovine endothelial cells and HUVEC and work well. It maybe pure luck, plasmid related, or some other unknown reason I had experience that one transfection just did not work with with lipofectamine 2000 or lipofectamine LTX. I guess you just need to keep trying until one will work for you.
Lipofection is not very effective in transfecting HUVEC that kind of slowing growing primary line. You may consider using lentiviral expression system. I actually tried infecting a few genes into HUVEC, it worked quite well. See if anyone at your institution you can ask for the materials and the technical support (culture hood for virus). That's not hard and it is not expensive, all you need is the expression, packaging, envelope vectors (available in Addgene) and the packaging 293T cells and any lipofection reagent for transfecting all these vectors into the 293Tcells.
Thanks for the answers so far!
Would also a normal electroporation work, if Amaxa is working? E.g. with a Biorad Gene pluser?
Elivira,
It is not always the transfectant agent which determines the efficiency of transfection but also many other factors such as the vector used to transfect the cells, the suggestion by Hong is really a great suggestion, another factor from my experience is that the gene itself.
good luck
During my PhD thesis I tried nearly everything in bringing plamid DNA into HUVEC... The only way it really worked with up to 20-30 % of efficiency was good old Calcium Phosphate precipitation method - of course cumbersome, because you have to establish buffers with different pHs, and the optimal pH is within a very narrow range. But once you have your optimal buffer, you will be a very lucky guy :)
Hi Elvira
what is the method that you used and worked well with your transfection in HUVEC?
thanks
The Targefect-HUVEC reagetn works well for transfectign all types of primary endothelial cells including HUVECs. You can view details at the following link
https://www.nature.com/protocolexchange/protocols/2096
HUVEC cells should be easy to transfect using cationic liposomes. If you have the necessary tags for HUVEC cells, then the complexes you make should have an easy time entering into the cells. The reagent below is not really a generic - it's specific to HUVEC cells - so see if it works for you.
https://altogen.com/product/huvec-transfection-reagent-human-umbilical-vein-endothelial-cells/
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