I ordered and produced a plasmid from Addgene containing PIK3CA and GFP (pHAGE-PIK3CA-C420R (item #116457).
I performed mini- and midi preparations and verified plamid production with a restriction enzyme digest and PCR of the GFP region.
In my experiments, I used this plasmids to produce lentiviruses via transfection of HEK293t cells (the protocol included pMD2G, psPAX2 and jetpei trasfection reagent). I used another plasmid which has worked well for us in the past, as positive control (psin-ef2 with gfp insertion).
Following the trasfection I looked at GFP flouresence in the HEK293t cells. I noticed very little GFP emission from the PIK3CA plasmid compared to other plasmid (psin-ef2 with gfp insertion) which underwent the same protocol.
(Gene maps for both plasmids are attached)
Any ideas on what to do to increase lentiviral production and GFP emission in the HEK293t?
You should follow the Lipofection technique for your plasmid transfection to 293t cell.
If I understand correctly, your control plasmid (which you have inserted a GFP gene into) has a marker for Puromycin selection. Presumably you add a drug selection to the cells after transfection. In this control plasmid the GFP expression is driven by the EF-1a promoter (I am guessing this is a relatively strong promoter). In you PI3KCA plasmid, in contrast, the GFP CDS is downstream of the PI3KCA CDS, and is using the IRES (internal ribosome entry site) to be translated. So while the same EF-1a promoter is driving GFP in both plasmids, I wonder if the IRES is relatively inefficient in the PI3KCA plasmid--and so the GFP emission is lower?
Also, why is there no Pur gene in the PI3KCA plasmid? What drug selection do you do for that plasmid?
I haven't read the original paper describing the PI3KCA plasmid, but perhaps that protein product is slightly toxic to cells so the copy number is lower? (I am just guessing).
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