Has anybody tried injecting mRNAs encoding GFP (or other fluorescent proteins) into the hermaphrodite germ-line? We have been injecting lacZ mRNAs as translational reporters, but i need an alternative method that does not involve killing the worm.
I made my mRNA using mSCRIPT mRNA kit from Epicenter. It states a final conc of about 60ug of mRNA can be obtained from their kit. I got around that amount on some samples but others were a little lower ???
The kit comes with 5’Capping and 3’ Poly ‘A’ tailing reagents. I used both linearised plasmids and PCR products with the T7 promoter as templates and both worked well.
I injected my mRNA’s between 500-1000ng/ul. This is high but I didnt see much eGFP under a dissecting scope. We now have a much higher resolution scope but I have stuck with the above concentrations as I know it works.
The number of animals seen with eGFP relates directly with how good your injections are. On a good day I get around 60-70? of my injections to show eGFP. I also noted I got eGFP expression in tissues I mistakenly hit eg the intestinal cells :-\
The only thing of note is we had our eGFP sythesised as a DNA sequence that was codon optimised for C. elegans. I dont know if this is something you would want to consider.