I have a GFP expressing transgenic line and I need to obtain a stable line.
I have used a UV cross linker (stratagene) and screened more than 100 F1 (couldn’t find more as the parents die) but I didn’t get any 100% F2 after cloning 6 animals from high transmitting’s F1’s. :’(
Can anyone give me any advice ?
Thank you very much
Hi,
You will need to pick many more F1s. I would recommend picking at least 300-400.
Good luck!
We soak the worms in trimethyl psoralen (TMP) prior to UV irradiation which enhances the UV effect. There’s a protocol on this page, but many of the 'micro’s have been switched to 'milli’s. It should be 50ug/ml final concentration, and I believe we add that to an equal volume of washed late-L4’s or very early adults. Let the worms mix with it. Then put them on the plate with no food to rest and to let the liquid soak in. Then you hit them for 300uJ and then add the food. We put the plate on a stand to get them a few inches closer to the light source. You’ll still need to pick a lot more F1’s (300 or so).
Using a Stratalinker we average 1 integrant every 150 animals picked, but still plan on picking 500 for any given array. It’s a numbers game, and not all arrays are equal. Also, some integrants express at lower levels than the originating array, presumably because the integrated piece was merely a fraction of the array, and you have lower copy number. So, always wise to hammer it hard and get multiple integrated lines.
sorry, may i know how to tell the difference of a stable integrated line from non-integrated line?
A non-integrated line is an extrachromosomal array, and is subject to mitotic and meiotic loss. Animals carrying the array will be mosaic (some cells have it, others don’t), and not all of their progeny will inherit the array. An integrated line has the transgene integrated into the genome, and can be mapped with respect to visible markers or SNPs. A transgene integrated into the genome behaves in a Mendelian fashion: all of the progeny of a homozygote will inherit it, and all of the cells of an animal carrying the transgene will contain the transgene (though not all will necessarily express it, of course).
For more information, you might look at the WormBook chapter; a lot of it is protocols, but there should be enough background information there if there’s any confusion.
I thought I should also mention pha-1 in the event tyrael’s question stemmed from using pha-1-based strains.
In practice, telling the difference is often straightforward: integrated = all progeny on a plate from a cloned mother will have the same phenotype; non-integrated = two different phenotypes (mutant + wild type).
However, when pha-1 is used as a transgene marker it won’t be so obvious. pha-1 mutation causes early lethality, so all the rescued animals [transgene + pha-1(+)] that grow up and reproduce are transgenic animals, whether integrated or non-integrated.
Hi Thanks for the prompt replies from chromosome_cowboy and HillelSchwartz
ya, actually i have tried the Gamma irradiation method to integrate a transgenic line with pha-1 marker… yes, like chromosome_cowboy told, i cant really tell which one is integrated strain (it’s a GFP transgenic strain), and everyone, well, looks still the same with the GFP intensity… thus my question came in the previous thread… is there any method, somehow, that i could verify it ?
I am not sure if this would be the best idea, but couldn’t you map the location of the integration?
As Snug says, you could try to map the transgene; if it’s linked to some marker, it’s integrated (depending on how you do your mapping; using certain mapping schemes, you could get the result that it appears to be linked to pha-1).
Also, the usual pha-1 mutation(s) used in making transgenes by pha-1 rescue are temperature-sensitive (so you can get viable animals to inject, before you upshift the temperature and look for rescue). So if you put the plates at the permissive temperature, you should be able to see which clones are capable of losing the transgene, and which can’t and are therefore candidates to be homozygous for an integrated transgene (or to contain gamma-induced enhancers of pha-1, I suppose).
In general, though, pha-1(+) would not be my co-injection marker of choice for doing an integration.