clonogenic assay for shRNA transfected cells - (Aug/29/2013 )

Dear all,

I'm planning to perform a clonogenic assay in order to evaluate the effect as tumor suppressor of a gene I'm studying.

The idea is to transfect cells with shRNA to generate stable transfectant, and then using the for a clonogenic assay. But in order to save time I was wondering if I could transfect cells with shRNA, treat them with the proper antibiotic to kill those that were non transfected, and then detach by trypsin and plate them again at the correct concentration (I think 1500 per well in a mw6). Somebody could tell me if this could be a good alternative that avoid me for waiting about one month and half before having some results?

In some protocols I showed people prefer to plate cell in a soft agar when cells do not form colonies when plated on plastic. Can somebody tell me if it is necessary to use a particular kind of agarose or the use of the normal agar for LB plating E coli could be good as well.

And in the caseof the soft agar method how is it possible to count cells within colonies. In many protocols you find that a colony is defined to consist of at least 50 cells. When cells growth as layer I think should be easy count them but how is it possible when the colonies are spheres?

Thanks in advance

Gianluca

It depends a bit on whether you want to make a monoclonal line or a polyclonal line.  If you don't really care about the levels of integrants and just want any stably transfected cells, then sure you can treat, pool (i.e. polyclonal cell line), and use those cells as your assay.  However, this will make it hard to assess a number of things, including growth rates and attachment properties, as some of the cells in the population will have more copies of your siRNA and this may alter how your results look.  When preparing a cell line it is VERY important that you characterize it before you use it - to ensure that it is what you think it is!

It is definitely better to select the cells before you start to do any work with them - that way you ensure that you a) have the transfected cells you want and b) can prepare stocks for perpetuation of the line - if you went straight into the clonogenic assay you would not be able to do this easily.

For clonogenic assays I use low melting point agarose rather than agar, as agar tends to be a bit hot when added to the cells, whereas LMP will stay molten at 37 for a while.

thanks bob1 for your replay.

And what's about your experience in counting cells withing a colony embedded within the agarose?

It is reasonably optically transparent so I haven't had any problems so far with a standard microscope.