summary: microparticle bombardment technology

Barth Grant grant at cuccfa.ccc.columbia.edu
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.

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 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
apparatus
spends most of its time lying idle.

-----------------




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