After transfection with CRISPR plasmids, puromycin selection and single seeding I hardly can see any surviving cells or they die soon in max 2 weeks. I tried gelatine coating, flat or round bottomed 96 well plates, adding amino acids to the growing medium and even trying different medium (L-15 medium and 0% CO2) and they die still. Has anyone managed to generate a knockout clone of these cells or has an advice regarding enhancing growth. The gene is not essential for surviving, I managed to knockout it in two other BrCa cells. Thanks in advance.
468 cells are notoriously hard to transfect (at least in my hands). Do your plasmids contain any fluorescent markers to determine if your plasmid got into the cells? Perhaps try optimizing transfection conditions (reagent, ul reagent, ug plasmid, cells per well, etc), and then have another shot at CRISPR modification.
Best of luck!
Thanks Craig. I have mCherry plasmids as indicator of transfection efficiency and actually the cells are quite well transfected. My question is more about growing the single cells after transfection and selection. Have you managed to generate clones from single 468 cells?
Hey Mostafa,
You could try to use conditioned medium. For example 70% of fresh growth medium and 30% of (sterile filtered) growth medium from a nearly confluent dish (ratios might be adjusted). The growth factors and supplements produced by our confluent cells may boost the survival rate of your clones. For me, this worked nice for Raw macrophages. Another note: Do you simple dilute your transfected cells into single wells or do you FACS sort them? FACS sorting might help to only select the transfected cells and reduce background, however the procedure might also increase your death rate.
Good luck!
Chris
Thank you Chris. I did not try using conditioned medium myself but a colleague of mine did and it did not work for her but still I can play around the ratios and give it another try!
I use puromycin for selection since transfected plasmids possess resistance gene against it when they don't express a fluorescent protein, then dilution for seeding single cells.
Hello Mostafa,
Is your puromycin resistant gene in Cas9 construct? Or did you add the puromycin R plasmid together? And does your puromycin R plasmid contain CRISPR-target sequence? (xxxxPAMxxxCMV-PuroxxxPAMxxx)
If your plasmid is All-in-One vector, Cas9 is going to cut the target until it loses the target sequences. And Cas9 will remain in genome. I think it's not good. When you add the PuroR plasmid separately, the cutting ratio and integrating ratio are independent. Good Luck. Random integration of drug resistance plasmid takes some time, so you can try to start selection after 72 hours or later. When you add the target seq both side of puro gene, it will be integrated to your target position. The 4th choice is homologous recombination. When you use Cas9 protein+synth RNA with target vector, only targeting unit will remain, which you can cut out later with Cre-loxP or FLIP system.
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