Replacing EGFP with RFP..but no expression! - (Jan/13/2008 )
We have a vector with EGFP under CMV promoter. We wanted to replace the EGFP by RFP as another gene needs to be cloned. This was done as follows:RFP(dsRed2) was cut between BamHI and NotI, cloned in to pcDNA3.1(+?) . taken out between NheI and XhoI and cloned in to target vector where EGFP was excised out between same sites. Cloning was confirmed by multiple sets of restriction digestions and DNA looks fine on gel. But when I do transfection on 293T cells, I do not see any red fluorescence! What could have gone wrong? Am not able to pin point at any problem at this time!
How long after transfection did you look at the cells?
We have noticed that Dsred takes extra time compared to GFP for proper fluorescence.
DsRed2 indeed takes longer to express than EGFP, it takes other exciation/emission filters (assuming you're using fluorescence microscopy to detect?) and is more sensitive to fixation (if you would have fixed your cells the decrease in intensity would be bigger than for EGFP).
so: how long after transfection did you look at your cells, did you fix them or not and what excitation/emission filters did you use (in both cases, EGFP and DsRed2). Any controls for your transfection?
I looked at cells after ~24 hrs generally at which EGFP shows up. I use fluorescence microscopy. The filter I use for EGFP is 488nm and for dsRed it is 558nm(excitation). The red filter is same which I use for PI. I observe the live cells which are of-course not fixed. May be next time I will wait for 48hrs.
Another point I forgot to ask: is it a fusion you're expressing or not? (DsRed2 is an obligate tetramer and not al fusion proteins will alow for the right tetramer conformation). Did you control with pCDNA3.1-DsRED2?
No the dsRed2 is not fused to other protein. I did not do control transfection with pcDNA dsred2 which I need to do.
There are now better genes for RFPs than DsRed2, such as mCherry, which do not have the tetramerization problems.
we have tried transfecting and infecting mcherry (only mcherry) and they have good fluorescence but they tend to aggregate in the cytoplasm. We have tried them in different cell lines and primary neurons, the same result, aggregates.
Have you sequenced the new DsRed2 construct?
I do not have the maps of the vectors, but are you sure that you have a Kozak sequence in front of the DsRed2 (maybe it is still there from the EGFP as it was excized with NheI)?
Does the DsRed2 has a stop codon or is the stop codon of pcDNA3.1 is used? In the later case, are you still in frame after using the mentioned RE sites? If you are out of frame, the translation won´t be stopped proberly and consequently the protein cannot be folded correctly (no fluorescence).
And have you checked your transfection:
Maybe the DNA prep ofthe DsRed2 is not ok. I would suggest to do a new prep (probably Endotox free) and try it again.
Hmm quite a good lesson learnt for me... we did transfection of the parental vector, subcloned vector(pcDNA) and the final lentiviral vector with dsRed2. For the first 2, fluorescence showed up as early as at 12 hrs. For lenti I waited for 48 hrs but even then it did not glow. After wasting enough time on this I just wanted to run the DNA on gel again to see that the lentiviral contructs (2 clones ) which I was using are both RECOMBINED and were running much lower when compared to empty vector! ... but still it is amusing as when we did restriction digestion for confirmation we got the expected bands!