summary: microparticle bombardment technology

Barth Grant grant at
Mon Apr 16 06:53:40 EST 2001

Hello All,

Here is a summary of information received on microparticle
bombardment technology.  Thanks for your help.


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

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).
animals die as bags, and are quite obvious among their
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
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
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
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
         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 days
at 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
spends most of its time lying idle.


Content-Type: text/x-vcard; charset=us-ascii;
Content-Transfer-Encoding: 7bit
Content-Description: Card for Barth Grant
Content-Disposition: attachment;

Attachment converted: MorganDuo:grant.vcf (TEXT/ttxt) (0001B46A)

More information about the Celegans mailing list