Transfection and Selection

Introduction

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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Question 1

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 … Continued

Question 2

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 … Continued

Question 3

What method do you think is the best for ease of use for transient transfection in HEK293?

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. … Continued

Question 4

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 … Continued

Question 5

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 … Continued