I am using 550A-1 plasmid. There is a EF1a-GFP-Puro, and a PGK-MCS in the plasmid. I want to put a tdTomato under the control of PGK promoter.
So if I use this lentivirus to infect cells, there will be both GFP and tdTomato expression in the infected cells. Will it be too toxic to the cells?
https://www.systembio.com/lentiviral-technology/expression-vectors/cdna/vector-maps
Hi Jizhou,
I think it will be not toxic but you can have a problem of expression with one of the two transgenes which will lightly expresses, and maybe the two... For a dual expression, this design is not very optimized. The best way is to use a 2A fusion sequence (promoter-GFP(2A)tomato), we use this design with rfp and luc and obtain a very high expression with of the two transgenes
Nicolas
Hi Nicolas,
Thank you. In my plasmid, the structure is: promoter-GFP-promoter-tdTomato-miRNA target sequence, so the expression of tomato is regulated by the miRNA target sequence, and the expression of GFP is independent and can server as an indicator of transduction. So can I still use the 2A?
Jizhou
ok, I understand better your experiment. In your experiental context, you can't use a 2A fusion according your purpose because you will have also a knowck down of the GFP due to the miRNA sequence. You told that you also have puro, is it GFP-2A-puro or GFP-IRES-Puro?
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