Anybody know how to increase the protein expression level of a compound strain?

Model Organism: Worms Methods & Reagents
1

I just made a compound strain by crossing a GFP marker into a transgenic strain expressing a protein X, the GFP was driven by the same promoter as the protein X.
I found the expression of protein X significantly decreased in the compound worms compared with the original worm.
Can anybody share some experience in terms of improving the protein expression?
Thanks in advance!

2

Weird and bad things can happen when you combine two separate and closely related transgenes, either by injecting into a transgenic line or by mating them to each other. Your best bets are (1) you can inject them together, instead of separately; or (2) you can minimize the shared sequence in the separate transgenes, which can mean avoiding the use of the same promoter and perhaps removing plasmid backbones prior to injection.

For more information, read up on “cosuppression” in elegans.

3

I second Hillel’s comments.

I assume that both transgenes are separate, integrated multicopy-arrays and they are both homozygous. The effect could be some kind of co-suppression, or maybe whatever is activating is in limiting concentrations. Does this effect occur with other independent lines of each of the reporters combined in one strain?

I cannot think of any quick fixes but you could try:

  1. Using a single-copy transgene for one or both reporters (although you might then see too little expression of either).
  2. Using a different promoter for one of the transgenes and/or making sure no mRNA sequences (e.g. 3’UTR) overlap.
  3. Co-injecting both transgenes simultaneously to make one transgene array. You would have to re-integrate it, but at least you could adjust the relative concentrations of the two reporter plasmids in the injection mix.
  4. Make a single transgene with a translational fusion of GFP with protein X.
  5. If you know a transcription factor that activates the promoter you are using, you could overexpress that from its own array, in the presence of the other two transgenes.

MM

4

I believe the phenomena is called ‘squelching’ where there is insufficient transcriptional factors and too many promoters, leading them to work less efficiently.

Just to chime in on Maduro’s #4, you could generate a one construct using a single promoter where the second coding sequence (gene B) has an SL2 trans-splice leader. This is usually done with the second gene being GFP so you know the GFP(+) cells also express gene A. There are several of these constructs floating around. Searching the internet for SL2-GFP reminded me of this paper using viral ribosomal skip sequences for the expression of multiple proteins from a single promoter:

http://www.genetics.org/content/196/3/605/suppl/DC1

-Kevin.

5

In terms of the co-suppression idea, we used rde-2 RNAi to improve expression of two pie-1 promoter driven integrated transgenes that became too dim when crossed together. This may not be appropriate for somatic co-suppression.

6

Thanks everybody for your input, I really appreciate it!