Hi All
I have been microinjecting to get transgenic worm lines of my interest. I always get 30-40% transmission rate of the extrachromoosmal array at best. Is there any way I can improve this rate? It would make my experiments a lot easier.
Thanks
hey joan,
that’s actually a decent % transmission rate for an array (yes, you can get higher, but also much lower). for 100% transmission you could integrate one of your arrays.
this is easy to do just using a standard UV transilluminator.
pick about 50 young adult transgenics (should have a row of eggs - L4s are not good!) onto on unseeded plate.
warm up your UV box for a couple of minutes.
place the plate (worms facing the UV - no lid!) on the UV box for about 5 seconds (depends on the box, so you might need to try
different timepoints).
distribute worms onto about 10 seeded plates.
single about 300 transgenic F2s and you should get an average of 3 integrated lines.
of course, now you have to backcross them since they have been mutagenized.
otherwise, there is always MoSCI or the gene gun if you want immediate integration.
good luck!
eric
I don’t know if it’s documented anyplace (and, heck, it may not even be true), but there is at least a persistent rumor that increasing the DNA concentration at injection can increase the frequency of transmission - although this is more a feature at the low end (preventing truly terrible transmission rates) than at the high end (I don’t think you’re ever going to get much beyond 2/3 transmission with an extrachromosomal array). It’s fairly common to bulk up your injection mix with DNA molecular weight markers or with a common plasmid such as Bluescript.
Another option - and it’s not actually one I encourage, but it is available - is to use a co-injection marker required for viability, such as a pha-1 rescuing construct. Mind you, this doesn’t actually do anything to your transmission frequency, but you won’t see the animals that lose the transgene.