A Discussion about Transfection and Clone Selection – Methods and Processes

We recently finished our Ask the Expert discussion on Transfection and Selection. The result was an interesting discussion about the issues surrounding transfection and clone selection including transfection of cell lines like HEK293 and dt40. 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. Discussion topics included electroporation, retroviral and chemical transfection, cloning media and transient transfection of viral vectors.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D. Dr. Fawcett has been in the biotechnology business for over 30 years. Trained as a biochemist he has held senior positions in both academics and industry and has been a mentor to many young scientists throughout his career. For the last 12 years Dr. Fawcett has been the Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland. He is also the Founder and Director of BioSciConcepts, a social venture of BTI that provides hands-on training for professional scientists in cell culture, baculovirus based expression, as well as topics such as molecular biology, PCR and real-time PCR. BioSciConcepts is an internationally recognized provider of expertise in the biological sciences and has provided consultation services to several small and large biotechnology companies.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert Transfection and Selection.

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.

The Answer:

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.

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.

The Answer:

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.

The Answer:

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.

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