Transformation of Dictyostelium discoideum with a particle gun
Contributed by Birgit Wetterauer and Hans-Ulrich Koop, published in
Wetterauer, Salger, Demel and Koop (2000).
Contact : Birgit Wetterauer
and Hans-Ulrich Koop
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Abstract
We transformed Dictyostelium discoideum using a simple home
made particle gun. The gun was built similar to the device described
by Finer et al. [1]. The main difference to commonly used
commercial systems is acceleration of the microparticles without a
macrocarrier, this greatly reduces the costs per shot.
Stable transformants were obtained at frequencies of up to 2500
clones/µg DNA. This is five times more than we achieved with the
same vector using electroporation protocols.
This page describes the protocol for transformation and gives a
description how to build a gun.
Introduction
How to contruct a particle
gun
Transformation procedure
Results
References
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Introduction
The principle of transformation with a particle gun is direct
mechanical transport of the DNA into the cell using metal beads as
carriers. Originally designed to deliver foreign genes across cell
walls of plant cells [2], this method has now been shown to
work for other systems too: various bacteria, yeasts, mammalian cell
lines were successfully used as targets [3]. Not only
unicellular systems but also leaves or entire animals (Drosophila
melanogaster, Caenorhabditis elegans and mice) can be transformed
[3, 4]. Applications of the method in DNA-mediated
immunotherapy and gene therapy are being explored [5-8]. The
so called "biolistic" method is capable of delivering DNA either to
the nucleus or into mitochondria [9] and chloroplasts
[10]. The general advantages of the method are short handling
time and high efficiency, however costs of the necessary equipment
can be regarded as a severe disadvantage. The simple and cost
efficient home made particle gun built by H.-U. Koop and P. Demel
overcomes this problem [4, 11].
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How
to construct the particle gun
Schematic drawing of the home made particle inflow gun used
in this study [see also 4, 11]. The scheme is not to scale.
The figure was prepared by Tim Golds.
To build a particle gun of your own you need the material listed
here
click here to view a larger version
of this image (623 kb)
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Transformation procedure
Material:
- 1. Gold particles of 1.5 to 3 µm (Aldrich, Steinheim,
Germany)
- 2. Tungsten particles of 0.7 µm (Biorad, München,
Germany) Comment 1
- 3. TE buffer
- 4. 0.3 M sodium-acetate
- 5. 96% ethanol
Procedure:
- Weigh 10 mg of the particles and suspend them in 200 µl
H2O
- Add 10-20 µg DNA in TE-buffer while mixing with a
vortex
- Precipitate DNA with 1/10 volume of 0.3 M sodium-acetate and
2.5 volumes of ethanol, incubate for 30 min at -20°C. The
particles will sediment.
- Resuspend the DNA coated particles in 400 µl absolute
ethanol; 13 to 20 µl of this suspension are used per single
delivery. The rest may be stored.
- Deposit the particle suspension on the metal sieve plate in
the "Swinney" type filter holder (see Figure) and mount it in the
vacuum chamber.
- To prevent evaporation of the ethanol in the vacuum, the
outlet of the filter holder is sealed with Parafilm fixed between
the male and the female part of a luer lock. This simple procedure
reduces shot-to-shot variation.
- We used 14 cm distance of the targets from the particle source
(less distance reduced survival of the cells, more could not be
tested) Comment 2
- We used a partial vacuum of -0.8 bar.
- Particles were accelerated by releasing pressurized helium (6
- 8 bar) by means of a solenoid valve (see Figure). The burst of
helium also ruptures the Parafilm seal.
- For stable transformation, vegetative AX2 cells at 5x106
cells/ml were concentrated to 5x10e7 cells/ml; 1x10e8 cells were
allowed to settle for 0.5 h in 6 cm petri dishes. This gives a
multilayer of cells. The medium was removed as completely as
possible, and cells were bombarded as described above. Cells were
allowed to recover for 3 h, and distributed on three 9 cm petri
dishes with HL5 medium containing 20 µg/ml G418 and incubated
for 1 week.
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Results
Most of our experiments were done with the plasmid p155d1-ecmA-Gal
[13]. This is a 12 kb vector with the 6 kb ecmA-Gal cassette
as an insert. The vector replicates extrachromosomally without
further requirements in the recipient strain.
Clones appeared after 5-7 days without further changes of the
medium. Stable transformants were found with an average efficiency of
1.5 ± 0.9x10e3 clones per µg DNA (see Table) 0.5x10e3
clones/µg using different electroporation protocols. Therefore,
particle gun transformation is about 5 times more efficient.
The procedure was highly efficient with the extrachromosomal
vector p155d1-ecmA-Gal (up to 2500 clones/µg DNA). With
integrating vectors the efficiency was roughly 10-fold lower, in
transient expression as well as in stable transformation. In
addition, there seem to be significant differences between different
integrating vectors.
Table:
Efficiency of transformation of Dictyostelium discoideum using a
particle inflow gun. In transient assays ß-Gal positive cells,
in the stable transformation resistant clones were scored as
transformation events. Means and standard deviations are given. In
all experiments 5x10e7 to 10e8 cells were treated. a) 0.65 µg,
b) 2.5 µgDNA per shot.
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1.
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Finer, J.J., Vain, P., Jones, M.W. and McMullen, M.D.
(1992). Development of the particle inflow gun for DNA
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2.
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Klein, T.M., Wolf, E.D., Wu, R. and Sanford, J.C. (1987).
High-velocity microprojectiles for delivery of nucleic acids
into living cells. Nature 327, 70.
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3.
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Klein, T.M., Arentzen, R., Lewis, P.A. and
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4.
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Wilm, T., Demel, P., Koop, H.U., Schnabel, H. and
Schnabel, R. (1999). Ballistic transformation of
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5.
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E.L., Sheehy, M.J. and Yang, N.S. (1999). Gene gun-mediated
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6.
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Sakai, T., Hisaeda, H., Nakano, Y., Ishikawa, H.,
Maekawa, Y., Ishii, K., Nitta, Y., Miyazaki, J. and Himeno,
K. (2000). Gene gun-mediated delivery of an interleukin-12
expression plasmid protects against infections with the
intracellular protozoan parasites Leishmania major and
Trypanosoma cruzi in mice. Immunology 99, 615-24.
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7.
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(1997). DNA vaccines. Annu Rev Immunol 15, 617-48.
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8.
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Sun, W.H., Burkholder, J.K., Sun, J., Culp, J., Turner,
J., Lu, X.G., Pugh, T.D., Ershler, W.B. and Yang, N.S.
(1995). In vivo cytokine gene transfer by gene gun reduces
tumor growth in mice. Proc Natl Acad Sci U S A 92,
2889-93.
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9.
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Johnston, S.A., Anziano, P.Q., Shark, K., Sanford, J.C.
and Butow, R.A. (1988). Mitochondrial transformation in
yeast by bombardment with microprojectiles. Science 240,
1538-41.
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10.
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Svab, Z., Hajdukievitz, P. and Maliga, P. (1990). Stable
transformation of plastids in higher plants. Proc Natl Acad
Sci USA 87, 8526-8530.
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11.
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Clapham, D., Demel, P., Elfstrand, M., Koop, H.-U.,
Sabala, I. and von Arnold, S. (2000). Gene transfer by
particle bombardment to embryogenic cultures of Picea abies
and the production of transgenic plants. Scandinavian
Journal of Forest Research 15, 151-160.
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12.
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Sanford, J.C., Smith, F.D. and Russell, J.A. (1993).
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bioloical applications. Methods of Enzymology 217,
483-509.
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13.
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Hughes, J.E., Kiyosawa, H., and Welker, D.L. (1994)
Plasmid maintenance functions encoded on Dictyostelium
discoideum nuclear plasmid Ddp1. Mol Cell Biol 14,
6117-6124
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