Hello, I am hoping someone can help us with an issue in growing and using EG4322 worms for transgenesis.
We have been using this strain for rescue based tansgenesis, both MosSCI and preparing EX-array transgenic lines for ~2 years without much problem.
However, we have recently encountered problems with the frequency of obtaining EX lines using both methods of transgenesis (using different mixes, different days, using clean/synchronized EG4322 worm stocks) over the last ~6 months. The transgene is not toxic-- we have previously made many EX lines using the same prep suggesting it is a problem with the background strain or our culture conditions. We typically obtain many rescued F1 worms but very few of these (even less than usual) give rise to EX lines.
Upon further inspection, we have noticed that the rescued worms have problems in developing to adult and usually perish around the L2-L3 stage.
The rescued and non-rescued ones often grow a molt and become stuck inside, leading to their death :’(. Very rarely do we get plates with F1s that mature into reproductive adults. The background EG4322 strains appears to have the same problem (lots of sick worms dying inside their molts).
We have always grown the worms in a box in a 25C temperature controlled incubator, or on the bench (also 25C).
We recently unthawed the frozen stocks made back when things were working well after receiving the strain. Unfortunately, these worms also have the molting problem suggesting it may be our culture conditions. However, switching between HB101 and OP50 does not seem to solve the problem. Also, N2 worms grow well on the same media, in the same box, conditions etc.
We have ordered the outcrossed version of this strain, EG6699 to replace our EG4322 in the meantime.
Are we missing something obvious? Does anyone have insights that may help us? THANK YOU!! ;D
I’d suggest raising your worms at lower temperature. In my experience unc-119 worms (even the outcrossed ones) are much sicker at 25 than at 15 or 20.
If the problem persists, we have CRISPR reagents for inserting transgenes at that locus in an N2 background, which eliminates the need to deal with Uncs. The plasmids will be at Addgene soon but if you are in a hurry, email me and I’ll send them to you.
Thanks a bunch, Dan. Unfortunately, it appears now the problem is not restricted to EG (unc) worms, but also N2 worms in the lab. I have grown them in temperature controlled incubators and at room temp (25C) on the bench. I see several (~5%) dead worms on the plate. Many of these are trapped inside their cuticle and they drag it around on the plates for sometime before starving. It must have to do with conditions related to the NGM plates we use. I just wonder if anyone else has had similar problems in the past, because as of now its going to be very difficult to narrow the problem to the source.
Any chance there’s something weird with your cholesterol? Worms need cholesterol to molt, and some of the genes that metabolize cholesterol in worms have molting defects and larval lethality (ie. let-767).