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Hi worm community,
The Glow Worms research stream of the Freshman Research Initiative at The University of Texas at Austin is a CURE course for undergraduate students to learn CRISPR via tagging of worm genes with GFP knock-in.
This semester we have 32 students tagging 32 genes. Next semester we would like to tag targets requested by the worm community. Our initial aim is to tag genes that would serve as excellent cellular or subcellular markers for distribution to the CGC and ultimately to you. We have funding for this project, so there is no cost.
Please feel free to submit requests here or to doonan [at] utexas [.edu] with a brief description of the potential high impact of tagging the gene. We look forward to hearing from the community! Thank you for your input!
Thanks a lot for doing such a great thing for both undergraduate students and the worm community!
Here, I recommend the icl-1 gene, which encodes the key bi-functional enzyme for glyoxylate shunt in C. elegans, an alternative energy metabolism pathway in relative to the Krebs cycle. The icl-1 gene is up-regulated in the daf-2 mutant and is associated with the longevity phenotype of daf-2 (Murphy et al., 2003 Nature). The icl-1 gene is also critical for the longevity phenotype of clk-1 mutant (Gallo et al., 2011 Mech Ageing Dev). The icl-1 gene is also involved in the fatty acid metabolism pathway (Goh et al., 2018 Aging Cell).
The reason for me to be interested in gene icl-1 is due to our most recent discovery that icl-1 is critical for the normal embryonic development of C. elegans in absence of mitochondrial SODs (superoxide dismutase). Eliminating mitochondrial SOD from either mice and flys leads to embryonic lethality, but mitochondrial SODs are not critical for C. elegans embryo unless the icl-1 gene is also missing. The ICL-1 mediated alternative energy pathway is clearly protective against mitochondrial stress, and play a significant role in longevity. Understanding more about the tissue specific regulation of icl-1 gene will provide more information for us to understand the role of energy metabolism in life and death.
The icl-1 gene stays in silence under normal condition, but it will be induced by stress, such as starvation or mitochondrial toxin, so it will be a good target for student to learn the cross talk between genes and environment.
If you have any related questions, please feel free to contact me. wangg5@dls.rutgers.edu
Do you think tagging icl-1 will be of general interest to the community or mainly to your own research? We ask because our goal is the former, to create strains that many labs will find useful. Once we have a list of candidates (including yours), we could poll the community.
There are alot of commonly used translation reporter strains out there that are from integrated extra-chromosomal arrays and not endogenously tagged. It would be a boon for the community to have these strains produced using a more precise technology (e.g. single copy expression using the endogenous promoter). I would contact the CGC (https://cgc.umn.edu/) and ask for a list of their most commonly requested reporter strains and use that list for the project.
@Rollins Thank you for the excellent suggestion! Do you (or does anyone else) have a sense of whether the most requested strains would also mean they are generally accepted as having non-artifact expression? Or perhaps more importantly, if they already label the desired organelle or cell as integrated array markers, would the endogenous tags be sort of redundant?
On the other hand, maybe it would be helpful just to have these re-tagged with better fluorophores like mNG or mScarlet?
Thanks again to everyone who has provided suggestions! Due to the remote, online learning environment resulting from Covid-19, our 2020 cohort of undergraduate students are researching the function/expression of suggested genes and preparing to collect reporter data from the CGC.
We aim to generate 50-100 strains per year as cohorts of students begin working in series and in parallel, so more suggestions would be welcomed! Thank you!
Hello, if you need any technical suggestion, please feel free to contact me at service@sunybiotech.com.
SunyBiotech provides high quality and efficient gene editing service in C. elegans using the CRISPR/Cas9 system, as well as related technical services, including Extra-chromosomal array, MosSCI, and plasmid construction.
We have successfully provided the services to nearly 200 labs around the world and generated more than 2500 transgenic lines.
I believe our experience will benefit your students.
Hi! I nominate her-1 , a gene specifically expressed in males, so anyone who is looking to identify males before the tail morphogenesis occur later in larval stages could use it.
This is awesome! I’ve been talking to a bunch of PIs about some kind of coordinated effort (and of course am still interested in that!) but this would be a great start. What about doing something thematically each semester? So if you do the sub-cellular markers in the fall, then switch to some other cell biological theme in the spring? and so on? Not sure if part of the course includes writing it up and submitting a paper - if there’s a theme, it would make that part easier. I could imagine doing TF families, signaling pathway components, cell cycle genes, actin regulators, etc. etc.
@Sevinc Ercan - Thank you for the great suggestion! Do any her-1 reporters already exist?
FYI another email request was lon-2 as an intestinal and hypodermal marker.
@david.matus - Thank you! Indeed, there are already others like Eric Hastie of the Sherwood lab at Duke and Sara Olsen at Pomona doing something similar (e.g. see https://www.ncbi.nlm.nih.gov/pubmed/31890079). At UT, we had the opportunity to try this at larger scale because of the well-established FRI program, which provides dedicated lab space, a budget, degree-relevant courses, and 1000+ students. Our longer-term goal/hope though is to collaborate with as many colleges and universities as possible to tag even more genes. We are hoping to be able to offer a tangible “Glow Worms in a Box” curriculum that others can adapt and customize to their own schools/programs. At UT, Glow Worms is a full year curriculum (spring, summer, fall) because that’s how FRI operates. We do 8 weeks of in silico CRISPR design, 8 weeks of cloning, 8 weeks of genetics/screening, and 8 weeks of microscopy/imaging. I assume this could be condensed into one semester though. And yes, we would love to be able to tag genes based on a theme! The possibilities are kind of endless, really, if we can get more programs involved. If you know of other PIs that are interested, discussing it further would be great!
This is a great idea! One suggestion (outside of specific target genes) would be to document and share successful sgRNA and HA designs so that others can easily make additional derivative tags (eg. Flp-ON, AID, various other FP’s) using SapTrap / SEC based protocols.
Thanks again to all that made suggestions. We followed-up on the suggestion of obtaining a list of the most requested CGC strains. We’ve tentatively decided that for the 2021 student cohort we will create endogenous tags for stress response genes and histone genes.
If you can help us curate the below CGC list (Google spreadsheet, public), indicating which genes/transgenes are involved in stress response and whether or not the gene would be a priority for tagging, we would greatly appreciate it! If you like, please also comment on whether the endogenous tag would serve to replace an existing transgene for the better.
We’ve gone about 250 strains deep into the CGC list and indicated the relevant gene and/or transgene. GFP strains are highlighted green. If we’ve made any mistakes, feel free to correct. Thank you for your help! We look forward to making strains for the community.
We’re about to begin tagging with our new 2021 cohort of students. We have an unusual amount of Neuroscience majors this year, so I’m wondering if anyone has a suggestion for a neuro-themed set of tags that would be widely useful to the community?
We’re also intending to tag stress response and rab genes, as previously suggested by the community (see discussion). Here’s the current list:
I am not sure if SKN-1 is on the list. If not, I think it will be very interesting to tag skn-1 gene at the C-terminus, preferably with a red fluorescence tag to avoid autofluorescence of the intestinal cells where it is mainly expressed.
As for hlh-30 that someone mentioned in the thread, we already made it and published it.
FYI, Henrik Bringmann has generated an NLP-29::mKate2 KI strain, making nlp-29 perhaps less of a priority.
As far as I know, there are no KI strains for the major MAPKs (PMK-1, PMK-2, PMK-3, JNK-1 etc). As some are involved in neuronal development, function and/or regeneration. As they are also important for multiple stress responses in different tissues, they could be suitable targets of general interest.
@Jonathan - Great, thanks for this info. It doesn’t seem to be archived at CGC yet, but we will just assume it will be. Thanks for the suggestion on MAPKs, too. That seems like a good set to move on to after rabs. We tagged mpk-1 last semester.
Btw, we are planning to tag the stress response genes with mScarlet so the students can compare the expression pattern to the existing GFP arrays or integrants. Figured this would be a useful analysis perfect for undergrads. If this seems short-sighted and mNeonGreen would be preferable, do let us know.
We’re generating knock-ins at a faster pace now and finally making good progress on the aims discussed previously in this thread, particularly creating the endogenous tags for the most widely used stress response transgenes. We’re planning to come up with a way to curate our progress in real time and publicly archive the details of how we made each strain (beyond what is curated at CGC) to facilitate using and genotyping them.
We’re hoping to survey the community again late this year to get new ideas for smallish subsets of tags (based on a theme) that would be widely useful to the community that we can tackle with the incoming 2022 cohort.
Hope you find the microPub useful and we hope to put forth our follow-up on improved plasmid cloning for the SEC method very soon!