I'm trying to make a stable cell line expressing two transgenes. I have no experience generating a stable line.
If I insert linearized plasmid with some selectable marker, I understand that could in theory generate a stable line, but I'm skeptical that the real-life efficiency of that is going to ever give me cells even the first time.
This is using the hacat cell line. I understand people have made stable cell lines.
The inserts are both too big to be put into lentivirus or retrovirus.
I understand piggybac would deliver a large enough payload. Would the efficiency for generating a stable cell line be better with piggybac, and could I do sequential or simultaneous inserts?
We have made hundreds of stably expressing cell lines. If you have two different genes you wish to express that are currently in two different plasmids, I would linearize them , transfect them together, and select for your resistance marker (neo or puro or whatever). Cells that integrate the plasmids will often integrate both plasmids, usually with multiple copies. Once you start getting colonies and you clone these out, you can test your individual lines for expression of the transgenes. If you screen enough of them, you will find some that express both proteins inserted. Of course, all of this assumes that expression of your transgenes is not lethal or inhibitory to cell growth. Expect to screen at least 20-50 cloned lines.
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