Is 3’UTR necessary for the expression of extra chromosomal arrays?
If we were to inject a classical (Gene of interest + GFP) construct like pPD95.75 and were to deliberately remove the 3’UTR (unc-54) sequence from the pPD95.75 plasmid… would the transgene still be expressed?
I would really like to know what happens if someone has already tried this or something similar before…
Thanks
it’s highly likely you will see GFP expression in at least some transgenic animals due to the miscellaneous DNA rearrangements that occur during array formation. Sometimes a fortuitous 3’ UTR will be generated.
So, the binary answer to your question is yes. The efficiency is going to depend on DNA concentration, coinjection marker etc.
Just out of curiosity, why do you deliberately want to remove the UTR? What’s the goal of the experiment? Like lmu1 said, it should express somewhere but you likely won’t get great expression from the array. Andy Fire has mentioned that one can omit a promoter from a construct and still get expression, but omitting a 3’UTR will lead to much lower expression. The 3’UTR has a huge impact on expression level and tissues in which the transgene is expressed.
Actually there is a particular reason why I want to do something like this.
So I am trying to insert in C.elegans, using CRISPR, a human ortholog of a gene. I’m using the approach published by D. Dickson. And using his vectors pDD282.
My current rescue construct is Left Arm–Ortholog–SL2–(FP-SEC)–Right Arm.
The problem that I’m having is that I’m unable to get any positive F1s carrying the extrachromal array. And I think the reason for this is that the human Ortholog is lethal in the high concentration.
And the Left Recom Arm actually has an active Promoter that belongs to the C. elegans gene, which would be disrupted after the ortholog is inserted.
So I was thinking to get rid of the let-858 utr sequence that is present after the GFP in pDD282 vector.
My having none or reduced expression of the ortholog as an extrachromsal array. I should be able to inject high concentration of the rescue plasmid (keeping the HR efficiency high).
And once I heat shock to remove the SEC I would get the expression from only the native promoter and utr as a single or double copy.
Of course I can change the around the sequence of the rescue plasmid, by having the ortholog just before the right arm, but I wanted to avoid extra cloning.
...I wanted to avoid extra cloning.
It’s going to be hard to remove the 3’UTR without additional cloning ;-).
Well removing the UTR (let-828) would be a lot easier … I just need to reverse PCR the vector leaving out the UTR part and then Auto-ligate the PCR product … of course I’ll be using Phosphorylated primers.
If you find that the UTR-less construct is still toxic and you do need to perform a more complex recloning, check out the SapTrap method from the Jorgensen lab (see http://www.ncbi.nlm.nih.gov/pubmed/26837755). It’s a modular system using Golden Gate assembly, and the current version of Dickinson constructs are compatible.
I think it may be worth considering what Sperm or Egg? suggested and go with the SapTrap system. It looks really efficient, modular, and easy to use. The let-858 sequence is to terminate transcription and insulate the SEC elements. Removing it could work, or it could also have unanticipated consequences, like readthrough from your strong promoter into sqt-1, and might affect your selection. If you try the site-directed mutagenesis to remove the let-858 UTR let us know how it goes. These SEC constructs are large and have repetitive sequences and transcriptional terminators, so might be challenging to linearize.
Are you trying to replace the endogenous gene with the ortholog? This might be one of those cases where recloning is a bit more work, but could save you a lot of hassle. If the construct is toxic, then you could put the GFP-SEC cassette first:
Left Arm-(FP-SEC)-SL2-Ortholog-Right Arm.
Thank you so much for the very helpful advice, I think I’ll just re-clone the thing as JordanWard suggested Left Arm-(FP-SEC)-SL2-Ortholog-Right Arm.
That was the original plan… I just wanted to avoid the extra long cloning, but it is definitely the better approach.
By the way are you the same Jordan Ward who made the pJW1219 Crispr vector? If you are then thank you for designing that …
I’ve had good luck with your more efficient crRNA targeting scaffold. ;D
And thanks Sperm and Egg for the SnapTrap article… I’ll be sure to implement that for new experiments. ;D
Cool, I think that’s a really wise move. In my experience, those experiments that seem like they should be easier can be a sinkhole and take way longer than starting again and re-cloning. I did indeed make pJW1219 and am happy to hear that it’s working 
Best of luck with your experiment!
Look, if you think toxicity is an issue, consider just making an array expressing your favorite gene, or failing to make such an array. If you can’t get arrays with it, consider introducing a frame shift in the middle, which should kill the toxicity. Your plans are clever, by why go to so much work when a quick validation of your model is simpler?
The general message is that endogenous 3’UTRs are precisely evolved, and we are learning that many genes use multiple 3’UTRs to control access of repressive elements, like microRNAs. Plus, the cell typically will employ nonsense-mediated decay to degrade mRNAs with abnormal 3’UTRs. Typically these are abnormally long UTRs, but I don’t think it’s that simple, and we really don’t understand the NMD mechanism of identifying abnormal transcripts.
There are also cute tricks to control toxic products.
Two require a ts mutant background:
But in the future I will likely use the auxin-inducible degron (AID) system, as applied by Abby Derberg’s lab. You tag your toxic protein with the small degron, and control its expression by adding/removing auxin, a cheap plant hormone that otherwise doesn’t alter C. elegans biology (or so we hope!) This also needs to be done in a special background, in this case an animal with a TIR1 expressing transgene, which has the added benefit of tissue-specificity if you need it. TIR1 is a co-factor required to mediate ubiquitylation and proteasomal degradation.
original methods paper:
http://www.ncbi.nlm.nih.gov/pubmed/19915560
Dernberg elegans application:
http://www.ncbi.nlm.nih.gov/pubmed/26552885
TIR1 transgenes at the CGC:
http://www.cgc.cbs.umn.edu/search.php?st=TIR1&field=all&exst=&exfield=all