Hi all. I’m trying to use the TU3595 strain (sid-1(pk3321) him-5(e1490) V; lin-15B(n744) X; uIs72 [pCFJ90(myo-2p::mCherry) + unc-119p::sid-1 + mec-18p::mec-18::GFP]) to see if I can determine tissue autonomy when knocking down putative axonal migration genes. Has anyone used these strains sucessfully for this purpose. My positive control isn’t showing anything and my test gene isn’t either. I will do some work to determine if my positive control clone is okay, but I’m curious if others have used this before. I’m particularly interested in knowing if the sid-1 rescue ‘sets in’ early enough. The original paper doesn’t look at migrations, but instead looks at mec adult phenotypes.
I’ve always wondered how feeding-based RNAi works in a sid-1(-) background with only unc-119::SID-1 to restore function. Maybe the thing to try is to make some neural stem-loop type constructs to express dsRNA in the nervous system in this strain.
Alternatively, if the genes you are interested in are restricted to axons in their expression, you could make a CRISPR/Cas9-induced knockout and restore function to a subset of neurons using neural promoters.
Just some ideas.
MM
Thanks for the response. I have trouble picturing the mechanism too. My problem is a gene that causes embryonic arrest, so I’m trying to get around that.
Hi, I tried it with an essential gene and it did not work in my hands. So I used an alternative approach that we developed (Esposito et al., Gene, 2007).
Let me know if I can be of any help with it.
Elia
Building on Morris’ response, you could try to go for a tissue-specific knockdown (if you have promoters that express specifically in your neurons of interest):
-somatic CRISPR editing. Instead of making a germline mutation and rescuing expression in the neurons, you could also express Cas9 under a tissue-specific promoter. Editing efficiency in somatic cells appears much higher then in the germline (http://www.ncbi.nlm.nih.gov/pubmed/25155554). For five genes they tested, they observed 86-97% penetrance of a known recessive phenotype; one other gene was less effective (20% penetrance)
-tissue-specific degradation. We just had a paper come out in Development describing an auxin-inducuble degron (http://www.ncbi.nlm.nih.gov/pubmed/26552885) By expressing a plant F-box protein (either ubiquitously or tissue-specifically) and tagging a protein with a 44 amino acid degron sequence, one can get rapid (20-40 min) degradation of the tagged protein. This degradation is completely dependent on exposure to a plant hormone (auxin). This approach has worked really well on the two essential genes I tested. And Liangyu, the lead author, had good success with germline specific knockdown of the dynein heavy chain, DHC-1.
The caveat of these two approaches is that they would not be as high throughput as an RNAi approach to screen putative axon migration genes.
The crispr cas 9 references are particularly helpful. Thanks so much!