RE protoplast transformation
altmann at de.dbp.mpg.mpimg-berlin
Fri Jul 10 08:06:22 EST 1992
we are using transient expression in Arabidopsis protoplasts routinely in
the lab. We are using PEG-mediated DNA transfer according to Damm et al.
(1989) Mol. Gen. Genet. 217, 6-12 principally only omitting the alginate
embedding necessary for regeneration. The complete protocol is being published
in Methods in Arabidopsis Research, C. Koncz, N.-H. Chua, J. Schell eds.
In brief, mesophyll protoplasts are isolated from leaves of axenically
grown plants. 500.000 protoplasts are usually incubated with 50-100 5g of
plasmid DNA (eg. pRT102GUS) in the presence of 20% w/w PEG. After washing
off the PEG the protoplasts are cultured in liquid medium for 2 to 3 days
to reach maximal GUS expression.
By T-DNA mediated transformation we also generated transgenic Arabidopsis
plants ecotype C24 carrying a p35S-GUS fusion which we can send to you.
One comment to your problem to detect transformed tumor cells: Vancenneyt
et al.in our lab constructed an intron-containing p35S-GUS gene that
allows detection of transformed cells very early after Agrobacterium-
infection (Mol. Gen. Genet. 220, 245-250, 1990). This construct worked well
in Arabidopsis and may also be useful in your case.
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