Can introduce a inverted polyA signal flanked by Flex in intron to generate conditional KO animal?
wYE
Dear All:
I just came up with a design not sure if it could work. Would really appreciate
some input.
I am trying to simplify the process of generating a conditional KO mice in my system by designing a universal stop cassette.
Instead of inserting 2 loxPs in different locations/introns of the gene, I am planning on to insert an inverted polyA signal (say SV40 early polyA) flanked by Flex in one of the introns.
In the presence of Cre, the inverted polyA will be 'corrected', which should result in transcription termination in this intron and generating a 3’ truncated transcript.
Does anyone know a good flex combination that are proven to work? (Which version of loxP works the best for this kind of purpose, I found too many possible combinations online and got confused)
And most importantly, would it be practical to introduce such casette(probably ~ 350bp long) by HDR after DSB mediated by RNA? In traditional homology recombination I have done much larger cassette but in that case a little loss of efficiency was not a concern, we had to pick hundreds of clones anyway.
Thank you very much!
Peppe
Miranda Wilson
Wow, this is a really interesting idea. I have been trying to put in two loxP sites around my exon but this is much better - only need one inserted sequence. I suppose the main downside is that any regulatory stuff in the 3' UTR will also be lost, while normal flox strategy should preserve the mRNA.
Peppe, can you share some more details on the mouse lines you mentioned?
Peppe
after reading wYE's post I realized I misunderstood it. He's willing to make a WT>KO transition, while I was referring to a KO>conditionalWT. Sorry about that. However, I'm still interested in both options.
The mice I was referring to have a transcriptional blocker on aspecific genes and the CRE can operate the removal of the cassette and restore gene expression in CRE+ cells http://jaxmice.jax.org/strain/006414.html. With this approach my concerns were about the insert size.
Did you manage to correctly flank your exon?
P
Miranda Wilson
No, I haven't really been able to flank the exon. My donor template has a traditional design with homology arms, upstream antibiotic cassette, and the flanked exon to replace the endogenous exon. My CRISPR nicking pair targets the exon. However, the exon in the donor is acting as a homology arm, so in most clones only the loxP nearest the antibiotic cassette is inserted. The few clones with both loxP sites I've generated also have exon mutations at the CRISPR cut site(s).
I was just trying to redesign my strategy (with CRISPRs cutting exactly where I want the loxP, adding the loxPs one by one if necessary) when I saw yours and wYE's posts. That mouse's transcriptional blocker is indeed a huge sequence! I see the Cre'd mice still show a phenotype - presumably having all those backwards cassettes and sequences in the intron affects transcription. A smaller polyA sequence only, as wYE suggested, should be less likely to mess things up.
wYE
wYE
wYE
Thanks for your reply!
Giuseppe D'Agostino
P
--
You received this message because you are subscribed to a topic in the Google Groups "Genome Engineering using CRISPR/Cas Systems" group.
To unsubscribe from this topic, visit https://groups.google.com/d/topic/crispr/By32riIchD8/unsubscribe.
To unsubscribe from this group and all its topics, send an email to crispr+un...@googlegroups.com.
For more options, visit https://groups.google.com/d/optout.
Giuseppe D'Agostino
Miranda Wilson
Not sure which would be the best polyA sequence, probably any of the ones used in expression vectors would be fine. Though I would be careful using SV40 since the full length polyA sequence has function in both orientations (early/late).
Most likely I'll design the pieces for the repair template and get the whole thing synthesised as a gBlock.