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Semi solid agar selection of stable transfectant - antibiotic?? - (Jan/23/2011 )

Hi Everyone,

I have just created some transfectants by electroporation and need to select single cells. I have tried cloning by limiting dilution, however the cells all died as they must need their friends around. I thought I would give selection via semi solid media a try.. What I am wondering is do I need to add my selective antibiotic to the agar/media? As I don't want to grow colonies which do not have the gene of interest..
Any help would be appreciated..

Cheers,

Katie :)

-k8e-

Hi
I also use to face similar problem, as many cell lines have density dependant growth properties. what i do is after electroporation, i give a medium change after overnight culture, after a brif wash with medium. until first 48 hrs i do not select using antibiotics. end of 48 hrs goes to selection which is continued for 2 days with medium change every 24hrs. by end of this time several non-transfomant cells die. at this point i introduce a low count of negative cells (wild type cells) of the same culture and contiue growing them for 5-7 days. due to this density some of the tansformaing cells survie and multiply for 2-5 or more cells. depend on the growth i again impart the antibiotic selection pressure just before 2-3days of colony picking. this has helped me to generate several stable clones. inaddtion you can introudce ROCKi after first selection process helps to hold up dying cells.
alternatively, one can grow the electroporated cells on inativated feeders (fibroblast ) but before hand you have to check wether your cell line goes well with coculture


k8e on Mon Jan 24 07:34:51 2011 said:


Hi Everyone,

I have just created some transfectants by electroporation and need to select single cells. I have tried cloning by limiting dilution, however the cells all died as they must need their friends around. I thought I would give selection via semi solid media a try.. What I am wondering is do I need to add my selective antibiotic to the agar/media? As I don't want to grow colonies which do not have the gene of interest..
Any help would be appreciated..

Cheers,

Katie :)

-vitalgene-

Hi!
Thanks for the advice.. Ok that sounds like a plan.. I do know there is about 20-30% positive cells in my flask cultures at the moment.. As I checked gene expression on flow.. Cool.. well I think i'll give the semi solid a go, and add the antibiotics like you suggest a few days before colony picking and that should allow positive ones only.. I also have some feeder cells so it may be worth giving those a try too..

Thanks for the advice,

Katie :D

vitalgene on Mon Jan 24 14:20:49 2011 said:


Hi
I also use to face similar problem, as many cell lines have density dependant growth properties. what i do is after electroporation, i give a medium change after overnight culture, after a brif wash with medium. until first 48 hrs i do not select using antibiotics. end of 48 hrs goes to selection which is continued for 2 days with medium change every 24hrs. by end of this time several non-transfomant cells die. at this point i introduce a low count of negative cells (wild type cells) of the same culture and contiue growing them for 5-7 days. due to this density some of the tansformaing cells survie and multiply for 2-5 or more cells. depend on the growth i again impart the antibiotic selection pressure just before 2-3days of colony picking. this has helped me to generate several stable clones. inaddtion you can introudce ROCKi after first selection process helps to hold up dying cells.
alternatively, one can grow the electroporated cells on inativated feeders (fibroblast ) but before hand you have to check wether your cell line goes well with coculture


k8e on Mon Jan 24 07:34:51 2011 said:


Hi Everyone,

I have just created some transfectants by electroporation and need to select single cells. I have tried cloning by limiting dilution, however the cells all died as they must need their friends around. I thought I would give selection via semi solid media a try.. What I am wondering is do I need to add my selective antibiotic to the agar/media? As I don't want to grow colonies which do not have the gene of interest..
Any help would be appreciated..

Cheers,

Katie :)

-k8e-

I use semi-solid media to do clonal selection--I used the Genetix clonal media I think--it has some additive to help CHO cells grow but you can get it without that. Anyway, the media comes 2x in a 100ml bottle and you add 2x media plus your selection agents and cells to make up the other 50ml. When I did it I had about 8 96-well plates made from this and after 11 days I could look under the microscope and find single colonies. I had a lot of single colonies and it worked really well. Stem Cell technologies has a similar product as well, but it costs like twice as much.

-briguy7-