Hi,
I am trying to integrate lines with extrachromosomal arrays using a UV strata linker. All available protocols ask me to irradiate the worms at 300*100 micro joules/cm^2. None of them tell me how many seconds/minutes I should expose the worms. Can anyone help me out in this please?
Thanks
Sudhanva
When I’ve integrated lines I just stuck my plates in the Stratalinker and pressed “energy mode” and then 300 and start. There was no need to program time/exposure.
Out of curiosity, how many worms do you put in the stratalinker? And do you use TMP as well, or just the UV?
I’ve always put 30-40 L4 transgenics on multiple plates.
A few days before I’m ready to integrate, I’ll just pick some N2 L4s and throw them in the Stratalinker to test if the UV bulb is okay (they should look a little sick).
I’ve only used just UV and it seems to work ok
What I’ve done for our stratalinker was a sterility curve of doses to find a sweet spot where occasionally I got a sterile plate, but most of the plates have decent fecundity. You don’t want densely populated plates, or that argues you aren’t hitting them hard enough. Currently with our Stratalinker we are doing 10-12 mJ. But I emphasize that machines can differ, so an empirical approach is ideal. Shouldn’t take too long, but make sure to do multiple plates per dose, since results vary significantly from plate to plate.
Also, I use no TMP, so no hazardous waste. I generally pick 5-6 array-bearing L4s per plate, irradiate, let the plate starve. Later (no more than a month) chunk a given plate to 4 new spotted plates, then from each of those chunks pick 5 array bearing animals (L4 or adult) singly to plates (total: 20 plates from 4 chunks per single original irradiated plate). From these picked plates, screen for 100% transgenics. Using this protocol, I get ~ one integrant per 140 animals screened (or 7 plates irradiated).
I usually do these in groups of 100-200, which is only 5-10 irradiated plates. A nice thing about this protocol is that you can irradiate plates, starve them, then chunk in convenience with your work load. Just don’t let them starve too long, or recovery goes down. Also I’m told, but haven’t tested, that starvation is critical.
Just did this last week and got 3 integrants from 300 plates screened (15 UV irradiated plates). These were done at a dose of 12 mJ, 6 L4s per irradiation plate.
Also, not a bad idea to check dose of fluorescent marker expression in resulting integrants. Sometimes expression is comparable to the original extrachromosomal array, sometimes much weaker.
One more question - what’s the highest penetrance array you’ve had success integrating in this manner? For gamma-irradiation I’ve heard 10-30% penetrant is ideal, so that way you can easily tell integrated from non-integrated. I presume it’s the same for this technique but was curious.
steve
Indeed, I’ve been told the same thing but I never tested this idea myself
Yup, it’s the same (good to start with a low penetrance array in order to distinguish integrated from non-integrated). I’ve had success from up to about 50-60% penetrance.
The apparent advantage of low-penetrance arrays is that you can actually identify heterozygous integrants. The integrants have Mendelian inheritance, and thus as hets are present in 75% of the population, which sticks out f you’re working with arrays with 10-30% transmission. Otherwise, you rely on identifying homozygotes, which of course are only 1/3 of the animals harboring an integrated transgene.
In practice this trick isn’t as useful as it sounds. At low frequency arrays will change their stability, so I’ve gotten a lot of false positives that I’ve then picked out 12-fold to try to homozygose. Not fun. So while that trick sounds great, I’d don’t think it confers much of a practical advantage in reducing the amount of work required.
So higher transmitting arrays actually work just fine. As for a maximum limit, I look for <80%. Anything from 10-80% will work. Just scan each plate looking for non-transgenics, and go with the plates where there are none. Then pick out a few progeny to confirm. It is easier to, say, rescue a visible marker, but we’ve done this with only GFP-marked arrays. My personal favorite for subsequent integration is generating an array rescuing dpy-20(e1362) (PS99 at the CGC). That way I just scan a plate and toss it the moment I see a Dpy. Makes screening at the end faster, and then I quickly outcross the integrant base on a GFP marker, thus losing the Dpy. But obviously it depends on each application.
thanks guys!
Picky technical question: Did you irradiate each plate singly or did you put multiple plates? And do these plates have food, or do you transfer irradiated worms to plates with food after treatment?
Irradiated on food, multiple plates at once. The UV lamps are in a bank at the top, so dose in the middle is probably fairly uniform. But I wouldn’t pack plates up to the edges of the apparatus.
Not that it matters. 12 mJ takes ~10 seconds on our machine, so one could zap a bunch serially. {Warning: your machine should have safety interlocks to avoid exposure, but fer Pete’s sake avoid these lamps when on; they really crank the UV and will burn the crap out of you if your machine isn’t adequately protected.}
On thinking about it more, I want to emphasize: 1 in 150 is a mean. We’ve had to go up to a thousand to get one before, though we usually get one in a few hundred. Not sure what variables dictate this. But people should know that sometimes brute force is needed. Don’t assume you can just do a couple hundred and succeed. Elbow grease sometimes needed.
Also, there was one weird event this summer: I generated 3 arrays, then an undergrad worked to integrate that which transmitted at lowest % (~10%). He screened through about 800 without getting an integrant. So after he was gone, I screened another 1000 myself and also struck out. I then screened the other two arrays (with transmission in the 60-80% range), and got integrants no problem (those I just finished, 1 in 100 mean). I have no idea what was wrong with that single array so that we couldn’t get integrants, but worth knowing about. A mystery event, and I welcome comments if people have seen something similar.
Some relevant and possibly helpful notes compiled by Morris Maduro can be found here: http://www.faculty.ucr.edu/~mmaduro/int.html#uv
For example, it is suggested by Eric Moss that, in his experience, bacteria acted like sunscreen…
“Make sure there is absolutely no E. coli around. I tried several times to do a dose-response curve with food on the plate and got wildly different results each time. I finally got repeatable results when I washed the worms out of all the bacteria and put them down on an unseeded plate. The bacteria absorb the UV – they must act like sunscreen!” (Eric Moss)
Comment (M. Maduro): This is a great suggestion! I had assumed that as long as the plate lids were removed, the UV treatment itself would be fine. This is an important observation about UV, and may explain why initial attempts were not successful.