Hope you can help me with this issue… I’ve been microinjecting N2 worms with a construct containing the promoter region of my gene of interest upstream the GFP (using the plasmid ppD95.75). . I’m using MyoD::RFP as a microinjection marker. I’ve microinjected lots of worms and I always have lots of red F1 worms (some of them passing to the next generation). However, until now, I only have had one green worm (also red, of course). And the green expression didn’t pass to the next generation… :’(
-Is that normal? What is going on? I’ve been trying several things to obtain again my green worm, such as microinject exactly with the same DNA mix, change DNA concentrations, use younger N2… I don’t know what else can I do!
We know that the promoter length we’re using is enough to drive the expression of the gene since it rescues the lethality mutant strain with a deletion for this gene.
i guess you sequenced the promoter/gfp junction and it is ok?
do you have a decent kozak consensus sequence upstream of the ATG for GFP? (probably, if you cloned into a standard site in ppD95.75).
when you made your rescuing construct did you use a cDNA for your gene? i’m guessing probably not. in that case there might be important enhancer sequences in one of the introns, particularly the early ones. they could also possibly be in one of the exons, although i don’t know how common that is.
if you want to assay things quickly and dirtily you can do PCR to amplify candidate fragments, include about 25 bp of homology to a cut site in pPD95.75 in your downstream PCR primer, and coinject with cleaved pPD95.75. these will recombine in vivo at high efficiency (see kemp et al. 2007. “In vivo construction…”). use a myo2 promoter fragment or something as a control.
So, yes, I’ve sequenced the promoter/GFP junction. But, what do you mean if it’s ok? The promoter is cloned in the MCS of the pPD95.75 (in XbaI- BamHI exactly), before the artificial intron of the vector. And yes, it has a Kozak sequence just before GFP ATG (GAAAAAATG).
Your guess was right, the rescuing construct was made from the gDNA, not the cDNA. I also thought that there might be some regulatory regions in the introns, but the early introns are really small. Do you think that they can contain regulatory regions anyway? On the other hand, the third exon (there are nine) is really huge, so maybe you are right and the regulatory regions can also be in this exon.
I think I’ll try what you suggested doing the PCR of the fragment containing the three early exons and introns.
Given that you have a rescuing genomic clone, one obvious thing you could do is to clone the gfp open reading frame into the genomic clone, to get a GFP-marked rescue construct; I was able to do this successfully for a gene from which I was getting confusing results trying to make a “transcriptional” reporter. Of course, for some genes this may not work at all (though persistence can help; it took me several tries to find a place in the gene where the gfp open reading frame would not disrupt rescuing activity and would give fluorescence), and for other genes the subcellular localization of the protein could impair efforts to identify the cells.
The other thing I would strongly recommend is that you make a “minigene” construct: get a full-length cDNA for your gene and use it to replace the gfp open reading frame in your transcriptional reporter. I found for my gene that a cDNA fused to a fair few kb of upstream sequence didn’t rescue, but when I tacked onto (or into) that upstream sequence a large intron from within my gene I could get cDNA rescue (position and orientation weren’t very important). Without some version of this test, you won’t know for sure that the elements you have driving gfp expression are sufficient to drive expression where and when your gene of interest is required.
yes, by “ok” i was just wondering if there could be a trivial problem with the junction.
i have to agree with hillel that things can get messy when you start adding portions of the ORF to GFP, i.e., you could wind up with something that is toxic or very unstable. if your gene product (assuming it encodes a protein) has discrete domains then that could help for picking places to make the cutoffs.
and then there is the annoying 3’ end of the gene, which can (but usually doesn’t) harbor important transcriptional regulatory sequences (but elements that govern stability and/or translation of the mRNA are not unusual). the most conservative bet is to use recombineering or PCR stitching (or Gibson cloning) to insert/attach GFP (or some other tag) to the intact genomic context. ideally this would also be on the chromosome, but in practice expression levels are often better from an array anyhow (unless you want germ line expression…).