I'd like to perform a stable transfection to human cancer cell lines. I performed the transient with pCVMTNT vector, but I want a stable expression of a protein, which I study.
The cell lines I'm working is the HCT-116 and T47D cell lines.
Liposomes work for both HCT116 (https://altogen.com/product/hct-116-transfection-reagent-colon-carcinoma/) and for breast cancer cell lines in general (like MCF-7: https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/). It takes us less than 4 weeks for the generation of stable cell lines, as this requires multiple rounds of selection to ensure a homogenous population. Selection markers are practically required for the generation of stable populations, but positive indicators like GFP won't work.
Hi Panagiotis,
any vector containing a mamallian selection marker should work if:
I) the cell line you are using is sensitive against the antibiotic
II) you are able to transfect the cells
III) your cloned gene is not toxic to the cells
IV) the promotor which drives your cloned gene is not silenced in your cell line
First you should determine the minimal concentartion you need to kill your untransfected cell with the antibiotic. Then transfect your cells (electroporation or lipofection). Wait for 2-3 days, expand your transfected cells on several dishes (if you want to isolaote single cell clones, they should be big enough to get nicely separeted colonies) and add the apropiate concentration of antibiotic. Wait until colonies are grown, then isolate your single cell clones and pool the rest.
If you do not succeed, then you can tranfect a linearized plasmid, this should enhance the integration rate.
All the best,
GAD
Hi Panagiotis，I usually used lentivirus expression vector, such as pLVX-IRES-Neo and pLL3.7, you can go to the addgene to find some relevant plasmids. Good luck to you!
think about integrating your construct at the aavs1 locus with cas9/crisp system..
see genocopia web site...
this way you can probe the function of you prot in any cell lines,
Liposomes work for both HCT116 (https://altogen.com/product/hct-116-transfection-reagent-colon-carcinoma/) and for breast cancer cell lines in general (like MCF-7: https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/). It takes us less than 4 weeks for the generation of stable cell lines, as this requires multiple rounds of selection to ensure a homogenous population. Selection markers are practically required for the generation of stable populations, but positive indicators like GFP won't work.
- Badges
- Science topic