Gene Gun Technology@elegansNet
Bombardment Technology - Barth Grant [email@example.com]
webcasted on 4.17.01 via firstname.lastname@example.org
Here is a summary of information received on microparticle bombardment technology. Thanks for your help. Barth
Perhaps you know by now, but the Praitis et al paper on this finally came out, in Genetics (157:1217-26). They did in fact cite the apparatus you mention.
Although we are still in the technical phase of this we have been successful generating several integrants. The success rate initially was only about 1 integrant out of 10 bombardments but is improving (last exp. was closer to 25%). Results of germline expression are still pending. We have been using the Biorad Biolistic PDS-1000 and I find it works very well. We have been following a protocol developed by Vida Pratis, et al in Judith Austin's lab.
I've used the Biolistic genegun for transformations. Specifically, I blasted spe-26(hc138ts); lin-2(e1309) animals with spe-26(+) DNA and a GFP marker (expressed in the embryo). Transformed animals die as bags, and are quite obvious among their sterile untransformed siblings that accumulate oocytes. I was able to obtain 6 fertile lines (from 20 blasted plates), several of which were likely integrants, but I could not detect the GFP in any. Guessing that these were low copy number events, since spe-26 rescue requires germline expression, and since I can easily see the GFP in standard high copy arrays. Perhaps a useful strategy for germline expression, but the jury is still out.
After I read Vida's MCWM abstract last year, I got ahold of her protocol and, with a lot of advice from a postdoc in a brain lab that had a Helios system, I adapted it for the Helios. The method worked fine; the only caveat is that I did not do much with it - and nothing for germline exoression - so I can only say that it does indeed generate integrants at fairly high efficiency. By relative level of GFP expression and insensitivity to tam-1, the transgenes I got are likely low copy number and not highly repetitive. Furthermore, most transgenics only showed the unc-119(+) marker, not the GFP I was co-transforming, which may suggest a distribution of copy number for the GFP reporter construct centered between zero and one (or may suggest damaged, inactive copies of the GFP reporter construct). As to markers, I was adapting Vida's protocol, and I liked the rationale behind unc-119, so I went with it. It worked well; it is a very strong Dpy Unc and moving worms are immediately obvious after say 12 daysat 20 degrees. I found that, at least using HB101, her 'Opti-gro' plates are dispensible. For which machine to use, I would recommend what I did: look for someone nearby that has a system you can use if you provide your own disposables, and find or develop a protocol appropriate to it. After all, the Helios system is $20,000 or so, and I would guess the Biolistic is also very expensive; also, even in a lab that uses it often, the apparatus spends most of its time lying idle.
celegans/bionet.celegans Newsgroup Archive; Fri 13 Nov 1998; Bryan.Koch@mcmail.vanderbilt.edu
We got a big NIH grant to develop a mammalian version of our
To keep within the rules of our moderated newsgroup/list I will
Created: 4/17/01; Updated:
03/04/2003; Question & Comments;
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