Am currently transfecting Atlantic salmon cell line with pCDNA3.1-nCas9n-Hyg plasmid and wish to enrich the transfected cells via hygromycin selection. Is this possible? Is there an efficient alternative?
Dear Datsomor,
In transient expression, the plasmid doesn't get integrated in the genome of cells, it remains as an extra-chromosomal DNA inside the cell. It is possible to provide antibiotic and select only transfected cells. Generally, expression of proteins start after 24 hours of transfection. So you might add the antibiotic 24 hr post transfection. But, these plasmids if don't possess a replication origin which would allow it to replicate it inside host cell, after few cell division, the cells will be cured off the plasmid. In presence of antibiotic selection, they'll die even when their mother cell was positively transfected. Also the resistance gene expression might vary depending on the number of plasmids/cell. I think, its better to try to increase the transfection efficiency rather than trying to enrich the number of transfected cells. If you have higher transfection efficiency then most of the cells in the pool will have your gene of interest.
Hope it helps!
Yes it is possible... but rarely done. Hygomycin is not your best bet for it. You need a selection antibioitcs that is very fast acting. We had some reasonnable success with blasticidin. Just transfect at day one (to get the resistance gene expressed), select at day two (to killl the cells that are not transfected) ; rinse and assay at day 3...at 72h your transgene of interest is still fairly present....at later time point everyting goes back to background...
By the way, be carreful with your commerial source of hygromycin. This antibiotics is prone to highly toxicy but low level contamination. So some batches may generate residual long term cycotoxity.... so it will be much harder to get clones...
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