Rescue experiments using CRISPR KO cells with px459 vector

I've made a CRISPR KO cell line using the px459 vector. I would now like to perform a rescue experiment where I stably re-express the gene in the KO cell line by using for instance a plasmid encoding the gene cDNA? I would like to confirm that the CAS9 and sgRNA from px459 are not integrated in the genome of the cells and therefor should not cleave any plasmid encoding for the gene. Tlhank you.


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Brown Lamiaover 3 years ago

I think px459 has a puromycin resistance cassette in it. Have you checked if your cells are puro resistant? That might indicate that the plasmid has stably integrated. You might just not worry about this at all and express a cDNA that has the sgRNA sequence mutated in it. That's how I would do it.

Pearl Satyr4 months ago

Would you please share the detailed knockout protocol using px459 plasmid?
Thank you

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