I would like to do dilution cloning in immortalized mouse embryonic fibroblast cell lines obtained by serial passaging (to be sure that I work with a uniform clone). However, I don't know whether this is feasible, as MEFs often require cell-cell interaction to grow. Do you have any experience with that? Do I need a special layer (agar, feeders...), or will they be OK with plastic? Or is it impossible to tell and it depends on the specific cell?
Thank you in advance for your answers.
It probably depends on the specific cell, like you say. Those escaping replicative senescence could have any number of mutations. You will likely end up with some MEF lines that have lost contact inhibition and grow 3-dimensionally and some that still grow 2-dimensionally. You'll have some with functional p53 and some without it. You might consider using something like E1A-Ras to transform the cells able to grow nice colonies. Alternatively, as you indicate you could grow colonies on an irradiated or mitomycin C treated MEF feeder layer. GlobalStem sells pretreated cells, for example: https://www.tebu-bio.com/brandsearch/222/GlobalStem.html
For your dilution cloning you can try to cultivate the cells in conditioned media. Simply take the culture media from your immortalized (polyclonal) MEF cell line (preferably when the culture is 50-70% confluent), filtrate it to get rid of the cell debris and mix it one to one with fresh media. Then use this mixture for the cultivation of the clonal cultures.
Thank you all for your answers. The cells surprised me in a good way, the dilution cloning is working fine - with and even without conditioned media.
Dear all,
I also have similar questions regarding limiting dilution on MEF. However, I have primary MEFs that have passage no. is 5-6. I would like to knockdown genes via lentiviral transduction in primary MEF and do limiting dilution for selection and pick out single colony. Does anyone perform this procedure before?
Thank you in advance!
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