I want to generate transgenics from gene bombardment with my GFP-tagged protein but do not want to further subclone.
I already have a strain with my GFP-tagged gene on an extrachromosomal array from microinjecting but would like very much to have a strain with less copies of the protein via the gene bombardment strategy.
Wormbook recommends using a unc-119 rescue to identify transgenics but that would mean more subcloning. Does any one know of another strategy?
Thanks!
It is not necessary to clone your reporter and the cotransformation marker onto the same plasmid. If you prepare the microparticles with both plasmids, you will find that a fair proportion of the transgenic lines express from both plasmids. So more subcloning is not strictly required.
For a list of some other cotransformation markers that have been used, and for references for cotransforming two transgenes on separate plasmids, I refer you to Vida Praitis’s review in Methods in Molecular Biology
I have tried to use other lines to generate transgenics but I have rarely gotten them to work, and have never gotten an integration event. One of the problems is that if the worms are not from an unc background, they will move out of the relatively narrow area that the gene gun will target very quickly. I solved this problem by keeping the plates on ice until it was time to bombard, but even with this bit of help I had a lot of trouble generating any useful lines with this method. I tried the spe-26 ts mutation mentioned in the Praitis Methods chapter the previous poster suggests, and I had a horrible time working with them. All of my constructs worked fine when I injected them, but when I tried to bombard them into the genome, I got zero rescue from about 30 tries at bombardment, and the ts mutation screening requires keeping them at 25, which led to the growth of some pretty funky smelling microbes on my plates. I think that if you want to do bombardment, the unc-119 background is the easiest way to go, primarily because the phenotype of transgenic worms will be fairly unambiguous and easy to see.
To avoid having to subclone a gene of interest into the unc-119 plasmid used for bombardments
we routinely linearize both plasmids with the same restriction enzyme, perform a ligation reaction
for one hour and then precipitate the DNA. The pellet is then resuspended in TE at a concentration
of one microgram/microliter and bound to beads as usual. The bombardment is carried out
using standard conditions. Our efficiency is comparable to that for single plasmid bombardments.
We have not estimated the copy number of integrated plasmids. More details can be supplied
on request.
The C. briggsae gene rescues well and is much more compact, about two and a half kbp. I’ve never used it but Andy Singson (Rutger’s, New Jersey) says it’s great. If you need a source plasmid for PCR let me know. (mmaduro@ucr.edu) - MM
I have also rescued unc-119(ed3) with the C. briggsae gene and it works well for me.
Via DNA recombineering rather than subcloning for unc-119 ,
another procedure for Gene Bombardment trangenics construction
by using unc-119 as a marker .
[url=http://[/http://www.biomedcentral.com/1471-213X/8/119url]
[url]][/http://www.biomedcentral.com/1471-213X/8/119url]
its step by step protocol avilable here