Dual fluorophore expression in mammalian systems

Probes & Prep
1

Hello!

I am doing a lot of live mammalian cell imaging with fluorescent proteins but I am struggling to express two fluorescent proteins in my system. One of which is an inducible FP and so I initially tried to optimize expression but that didn’t seem to help as assumed my cells were just stressed. I was just wondering if anyone else had experienced these issues/how they worked around it.

Thanks!

2

Dear Karthrine,

Are you transiently expressing both proteins? Are there any genes attached to it? Which fluorophores are you using?

I’ve seen cases where one construct is much more effectively transcribed and required titration down relative to the other. This can be inherit to the transcription efficiency or, stability of the mRNA, folding of protein or tight protein control mechanisms which prevent overexpression. It’s hard to figure out which cause it might be.

Over all I’ve always avoided transient expression and resorted to making ‘stable expressing’ lines as most constructs contain an antibiotic selection marker. Also I’ve had best success with using inducible Tetracycline for this and even done dual inducible work with Cumate system.

In my experience, best to use two different promoters in my case CMV and EF1a worked well. I’ve seen recombinations and epigenetic silencing of one of the two promoters over time. Lastly, I’ve also used uORFs to dose the expression of the construct. That I did based on this paper: https://doi.org/10.1073/pnas.1305590110.

Good luck, the imaging is the fun part, cloning less so!

Teo

1 Like
3

Hi Teo,

Thanks for replying! Using GFP and mApple on plX304 and plX403 vectors respectively. Have been trying to make a stably expressing line and both FPs have ~50kDa proteins attached. Will have a read of the cumate system and uORFs so thank you for that!

Katherine

4

Hi Katherine,

Thank you for the additional information. Over all it is best to optimize your current system, rather than switch to CumateR.

Are you making actual lentivirus out of these plasmids, or transfecting with lipo/electroporation?

Some things that pop into my mind:

  • Can your protein(s) of interest tolerante c-terminal tag; checking the plasmids I see -V5-6xHIS-tag. Could interfere with mRNA processing (e.g. wrongly localize for translation, etc) or protein folding/stability (e.g farnesyl, etc, modification of protein).
  • For transfecting/electroporation: it can help to linearize plasmids the plasmid must open for integrating, so best to control this process: find a unique restriction enzyme in backbone part that is only needed for bacterial amplification.
  • make sure the DNA prep is clean, nanodrop measures should show no RNA and protein contaminants. I found electroporation is sensitive to this.
  • Simplify the system: I was lucky to be able to use cell lines that already stably expressed TETR, so plasmids were simpler (I used pcDNA4 or 5)

I hope you can optimize the expression!

Teo

5

Hi Teo,

Thanks again for getting back to me, I am making lentivirus. I am going to try look for ways I can simplify the system as you suggested, thank you!

Best,

Katherine

6

this can be a challenge. I’ve had good success by making the cell line stable via selection, then introducing the second protein, rather than trying to do both tagged proteins simultaneously. Have done numerous pairs together this way.

7

Hi James thanks for replying. I have tried this also as it had worked ok for me previously with different pairs of proteins but with my two current POI is a real challenge to find double positives. I have also made both lines stable separately and then introduced the other protein and get similar results. Not sure if just specific to this protein pair