Hi all,
I have a plasmid with "LTR >> EF1alphacore promoter > protein1-T2A-tagBFP-P2A-BlasticidinResistance >> LTR". I made stable murine 3T3 cells, using Blasticidin 20µg/ml, which is rather high.
However, although they seem to survive 1 week of selection, and the control cells all died within 48hours, not all cells are blue. Rather, only 0.5-1% seem to be expressing tagBFP. Looking with FLOW by staining for the protein1 which is localised to the nucleus using a 3xNLS sequence showed a smilier pattern (using the correct perm buffers). I then sorted them for BFP+, and the next day the % of cells expressing tagBFP is again rather low.
How can this be explained? What should I change? At the moment, the tagBFP and the Blasticidin resistance still have their start methionine, so in theory, they might be translated even without a Kozak sequence? Should I do SDM to get rid of those?
Or do I have different protein half life, so with a rather low transcription not a lot of cells stay blue but still survive the blasticidin? Should I exchange the promoter to e.g. CMV?
Really looking forward to your answers/suggestions! :)
Best wishes,
Chris
CMV promoter should help improve your tagbfp expression, also check this paper for multi-gene design comprising 2A sequences:
https://www.nature.com/articles/s41598-017-02460-2
Hi Christoph,
as the 3 proteins are supposed to be translated from the same mRNA you should not have separated translation unless you have an Ires or a cryptic promoter or cryptic splicing sites somewhere... (may be checked by sequence analysis ?)
what can happen is that your protein of interrest and the BFP are destabilized (fonctionally or reduced 1/2 life) by the 10 aa of the 2A sequences that remain at their Cterminus ....
the level of detection of the protein could also be different; antibiotiuc resistance more sensitive than blue fluorescence?
last thought: by using high level of antibiotic resistance maybe you favour/select cells expressing a lot of bla and less BFP (as there is no selection pressure on BFP)
good luck with your experiments
Thank you both for your answers. We are now generating a new template with CMV instead of EF1alphacore promoter. In the meantime, we isolated genomic DNA from our stable cells, and run PCR on them with primers targeting different regions of the inserted plasmid fragment. By now the cells have been propagated under selection pressure for 6 weeks.
We detected the whole insert, including the HIVgag element, parts of the protein, the BFP and the BlasticidinR.
Didier Poncet is there a Webserver to analyse if there are any cryptic promoters present?
Best wishes,
Chris
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