Hello! In order to isolate a reporter line, NPCs were electroporated with a CRISPR construct. The cells grow well as polyclonal, but when cultivating them as single cells for isolation of monoclonal lines, they start to experience high cell death. Has anyone experienced a similar problem and have found a way to improve survival? Or would it be better to maybe edit stem cells and then different them in neural progenitors? Thanks :)
Yes because the regular concentration of selection antibiotic is too much for a single cell.
I make polyclonal stable cells first in selection to make sure editing is happened in polyclonal cells and then go for single cells without selection in 96 well plate--> 24 well plate, until it becomes a confluent well. Then trypsinize and change to 6 well plate with antibiotic for DNA screening. once confirmed the desired edit, then freeze/no need of antibiotic.it takes time.
Thanks Goodwin G. Jinesh :)
But I only do antibiotic selection with the polyclonal population. The single cells are not under selection.
Have you worked with cells that have a completely normal background? Or they have some kind of p53, PTEN mutation?
Hi Maira P. Almeida
I would suggest you to test the efficiency of CRISPR/Cas9 in polyclonal cells (Mixed population), if you have the desired edit, then you can propagate the cells to single cell cloning without antibiotics selection, and if you did not have the desired edit then I think the high death rate due to electroporation rather than Cas9
- I did generate stable KO cell lines before where they express a comparable level of P53 to WT .
I worked with Hep3B which has TP53-FXR2 fusion that we found out:in this paper.
Data Mutant p53s and Chromosome 19 microRNA Cluster Overexpressio...
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