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Has anyone experience in isolating single-cell clones of CHO transfected cell li

CHO cloning limiting dilution transfection

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#1 Debora

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Posted 09 July 2014 - 07:13 AM

Has anyone experience in isolating single-cell clones of CHO transfected cell line by limiting dilution?

We are interested in transfecting a CHO cell line to produce a recombinant protein.
We are planning to use the limiting dilution method in 96-well plates to select single-cell clones to be screened for expression and we are looking for a detailed protocol for this cell line. Specifically, we would also be interest to know the cloning efficiency with this cell line (i.e. the expected ratio between wells plated and clones obtained).


Edited by Debora, 09 July 2014 - 07:14 AM.


#2 sara.r

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Posted 10 July 2014 - 08:50 AM

hi 

What kind of CHO cells you are working with adherent or suspension? if you are working with serum containing medium the cloning efficiency will be higher compared to serum free culture although there are a few cloning media which can increase the cloning efficiency in  serum free cultures, I once did serial dilution for adherent CHO cells in serum containing DMEM and  I did not calculate the effecncy but I can say I had about 20 single clones per 96 well plate.



#3 Debora

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Posted 11 July 2014 - 03:47 AM

Hi Sara!

Thank you for your answer.

We are actually planning to work with a serum-free suspension culture-adapted cell line.

We'd like to use eletroporation or lipofection as transfection method. What method did you use?

Can you give me some references of your protocol? Is it adaptable to CHO cells in suspension?

 

Thanks



#4 sara.r

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Posted 11 July 2014 - 09:30 AM

Hi

since our cells were adherent used lipofectamine 2000 reagent which had the transfection efficiency of about 50-60%. our protocol was really simple, I prepared 10 fold serial dilution from a 105 cell/ml stock to get 100 cell/ml and 10 cell/ml dilutions so I divided each plate in two parts and add 100 micro liters of each dilution in each part so I had 10 cell and 1 cell /well in each half of the plate. I prepared 5 plates and put them in 15% FBS  DMEM/F12 medium for about 2-3 week. you can fill the outer wells of the plate with PBS I suppose this will reduce the evaporation rate. after 2-3 weeks I checked the plates and marked the ones with single clones and refill them with fresh medium.  

 

for FBS containing culture the rate of getting single cell clones is higher but if you are using serum free medium you can consider using specific cell cloning mediums, I have not used any of those but I read somewhere that their effects can be cell specific. 



#5 Debora

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Posted 12 July 2014 - 11:21 AM

Thank you so much for your kindness!

I'll try to get some information about cloning in serum-free medium,

in the meantime I hope that someone that had experience with CHO in suspension culture maybe will add further details.

thank you







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