Transfection is often used to transfer an expression plasmid into an animal cell with protein expression as the goal. There are multiple methods to accomplish this and many opportunities to optimize the process. Do you know the best growth conditions for your cells when they are going to be transfected? If you wanted to isolate clones during selection would you know the best way? This session is a great opportunity to ask your questions about transfection and selection and practical, applicable answers.
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I am at the point where I have to transfect my cells and I read about both electroporation and chemical transfections. I have a fairly large plasmid (20Kb) and I wanted your opinion about the best way to transfect my cells.
Great question to start this "ask the expert". Yes both electroporation and chemical transfection are the two most popular methods for moving DNA into cells. The advantage of electroporation is that you can get DNA into all cell types and it is especially good for eukaryotic cells and stem cells. It works by using a very short pulse of high-voltage passed through a cuvette containing cells in a special conductive medium. It is important to optimize to minimize cell death, but with optimization done, you might get close to 100% efficiency with certain cell types,but not all. Of course you need an electroporation device of some kind. In case you need one Neon by Life Technologies is a good choice since it is simple to use and set up. Chemical transfection using cationic lipids is probably what people use most at this time for transfection. It does not require special equipment. One of the possible drawbacks is thatt you can't transfect all cell types. Another potential problem is that there is not one cationic lipid that works on all cell types, so you do have to do some digging to learn what specific cationic lipid will work for your cell type. My suggestion is that you should go to something like the Life Technologies transfection collection on their website, there you will find recommendations for many different cell types and references. I will mention that regardless of which method you choose, follow the protocols and do not skip any steps, equilibrations of reagents into transfection medium is important. Also, remember that optimization is important for your cell type. The same cell type grown in different labs might transfect with a different efficiencies because of the history of the cells.
Do you have any recommendations for best practices for transient transfection production of viral vectors?
I guess he answer might depend on your goals. Are you trying to produce a recombinant protein or are you trying to make a virus. I have used Cellfectin II many times with a viral vector that is about 120 Kb in size with good success. In my case even low level transfection is okay since infected cells produced virus which infected other cells. If you are working with large sized DNA be sure to treat it very carefully, avoid freeze thaws and don't vortex or shear it. I hope this helps.
HEK293 cells are highly transfectable. The cells can grow as both adherent if using medium with serum or they can grow in suspension is using a serum free medium. If your cells are adherent my suggestion is to use Lipofectamine 2000, if you are growing your 293 cells in suspension FreeStyle, 293fectin are good choices.
I want to stably transfect dt40 cells with a tagged transmembrane protein. Would you recommand electroporation or retroviral transfection? constructs for both ways can be cloned in my lab.
I have not used these cells but I have read a lot about them. DT40 cells is a B cell line from avian leukosis virus induced bursal lymphoma in a white leghorn chicken.
From what I have read this is a difficult cell line to transfect. Both Lipofectamine and FuGene don't work very well most people seem to electroporate if they want to transfect. Electroporation seems to me the be the simplest choice and the bottom reference has electroporation conditions and so do other articles I looked at.
Buerstedde J.-M. and Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines Cell 67, 179-188.
I found this web site which happens to be a very good reference for growing and manipulating DT40 cells http://cwp.embo.org/pc10-17/docs/Sale.pdf
I am looking for a way to use the same media for cloning and expansion. Do you have any suggestions on a media or supplement that would help me not have to change media.
When you write cloning and expansion I assume you are cloning and expanding for making a stable transfectant. If that is so, of course you will need to add whatever your selection drug is to the what is to follow. I believe what you are getting at is the difficulty of cloning a cell since there are few cells in a well or in a dish. Cells like to be around other cells so I recommend using conditioned medium that you can make. Conditioned medium contains nutrition yet since it has had cells growing in it it also has other factors that will make your cells happy while you are cloning them. To make conditioned medium use the same cells that you are cloning (untransfected) plate them at about 20% confluency and let them grow overnight. The next morning, using sterile technique, remove the medium and centrifuge to remove debris. Then add 50% fresh medium and 50% conditioned medium when you clone your cells. You can use this until your cells begin to grow, Once the cells reach 30% confluency or so you can add media that is not conditioned. If you are doing a drug selection for stables be sure to do a killing curve so you know the dose and the length of time it will take to do the selection. I hope this helps.