Hi, I have GFP fusions under hsp 16.41 promoter’s regulation which were successfully expressed in N2 by co-microinjecting with rol-6 at 100 ng/ul each (circular DNA). However, when I diluted the constructs with hsp promoter to 1-5 ng/ul (rol-6 remained at 100 ng/ul), I can get stable transgenic rollers but none of them express GFP (using the same constructs as in first round). I wonder if this is common for this hsp promoter? That only high concentration should be used for expression under this promoter? Thanks.
hey hanting,
i use constructs like this often. are you heat shocking your worms enough to see the GFP? why dont you try genotyping them with GFP primers? i normally see expression best after a 4h 33C heatshock followed by a 2h 23C recovery.
odysseus
I think this is more of an issue with amount of GFP plasmid DNA, rather than any specific issue with the promoter.
You may have reached a floor effect where you can’t get any less expression with that little amount of DNA injected.
As you inject less and less plasmid, it seems you reach a point where a copy of the plasmid either gets incorporated with the
marker array (and expressed), or it doesn’t (stochastically). This seems to be about 1-5ng/µl, depending on the plasmid
and the amount of marker.