I have been attempting to transfect my MDCK cells for a while now. However, every time I attempt to do this, the cells have shown little to no reaction to the process.
I am using Lipofectamine 2000 reagent from life technologies. The cells are grown in DMEM and later in G418 for selection after the addition of lipofectamine. I have followed the protocol and used the 1:2 ratio for DNA to lipofectamine concentrations.
There has been less than the 5% transfection rate that most literature seem to indicate. There are barely 1 or 2 transfected cells (if I'm lucky) in an entire 35 mm plate. Can anyone please advise me on what may be going wrong and perhaps a few suggestions on how to make this work?
Try using an MDCK-specific transfection reagent (https://altogen.com/product/mdck-transfection-reagent-kidney-cells-ccl-34/) that should help you overcome barriers in efficiency. Because MDCK is not a human cell line (it's canine I believe) generic reagents will not be optimized for the particular cellular markers present on the cells.
Hi Divya, As you might have already noticed by now that MDCK cells are very robust. It is quiet hard to transfect these cells using Lipofectamine. My suggestion is that you use electroporation as an alternative. I have used Lonza unit for electroporating MDCK cells and have got about 90% transfection efficiency. All the best :)
Hey Shankar.
i haven't transfected MDCK before but changing your protocol or read troobleshooting might hepl optimized your result. Can you try this protocol by using this link: http://www.dojindo.com/Protocol/H357_MDCK.pdf. Good luck.
We have had good luck in transfecting MDCKs with Lipofecatime 2000 (10-15%) but if you are trying to make a stable cell line, have you consider using a retrovirus system? Even with a good transfection, very few cells will actually be stably expressing your gene of interest whereas, with retroviral systems, you can easily get 50% of your cells surviving selection.
Whats extremely important when transfection cells, MDCKs or any other cell type, it to make sure the cells are in log phase growth state before transfection. I set up transfections from a stock plate of happy growing cells and if my stock plate is not in a happy growth state, I pass the plate and wait for the cells to be growing.
Are protocol for transfecting MDCK is to play 1.3 x 10^5 cells in a 24 well plate and transfect the next day with 4ul of lipo 2000 and 0.8 ug of DNA.
Dear Divya,
I would definitively recommend an alternative: DreamFect Gold. It is indeed a lipid-based transfection reagent, but highly efficient and biodegradable (high viability). DreamFect Gold presents a high level of nucleic acid compaction that allows using less DNA for a better transfection efficiency.
you can find more information at : http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html.
Concerning MDCK, DreamFect Gold is really efficient for transfection and also co-transfection as demonstrated for example by Zhang Y., Biochem Biophys Res Commun. 2010 or Grabherr MG.,PLoS One. 2011. Please find the link to the publications in the following.
I would be really happy to send you sample to test, please do not hesitate to contact me at : [email protected] for any information,
good luck with your experiment,
best regards,
cedric
http://www.ncbi.nlm.nih.gov/pubmed/20026304
http://www.ncbi.nlm.nih.gov/pubmed/21625601
Dear Divya,
we tested several lipid-based transfection reagents but non of them worked well for MDCK. In contrast, electroporation is very efficient in MDCK and we use routinely a microporator devise (now Neon Transfection System, Life technologies).
Best wishes, Timo
I never worked with MDCK but I had a very good experience with LipoD293 from Signagen to transfect difficult cells.
good luck!
Try using an MDCK-specific transfection reagent (https://altogen.com/product/mdck-transfection-reagent-kidney-cells-ccl-34/) that should help you overcome barriers in efficiency. Because MDCK is not a human cell line (it's canine I believe) generic reagents will not be optimized for the particular cellular markers present on the cells.
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