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.

	assay	plasmid	particles	number of experiments	events per µg DNA
a)	transient	p155d1-ecmA-Gal	gold	3	55 ± 22
	transient	p155d1-ecmA-Gal	tungsten	3	0.7 ± 1.1
	stable	p155d1-ecmA-Gal	gold	6	1500 ± 900
	stable	p155d1-ecmA-Gal	tungsten	3	1500 ± 950
	stable	integrating: pBsr2	gold	2	200 ± 130
	stable	integrating: ecmA-Gal	gold	2	140 ± 80
b)	transient	p155d1-ecmA-Gal	tungsten	1	8.4

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REFERENCES

1.	Finer, J.J., Vain, P., Jones, M.W. and McMullen, M.D. (1992). Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11, 323-328.
2.	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.
3.	Klein, T.M., Arentzen, R., Lewis, P.A. and Fitzpatrick-McElligott, S. (1992). Transformation of microbes, plants and animals by particle bombardment. Biotechnology (N Y) 10, 286-91.
4.	Wilm, T., Demel, P., Koop, H.U., Schnabel, H. and Schnabel, R. (1999). Ballistic transformation of Caenorhabditis elegans. Gene 229, 31-5.
5.	Rakhmilevich, A.L., Timmins, J.G., Janssen, K., Pohlmann, E.L., Sheehy, M.J. and Yang, N.S. (1999). Gene gun-mediated IL-12 gene therapy induces antitumor effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy. J Immunother 22, 135-44.
6.	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.
7.	Donnelly, J.J., Ulmer, J.B., Shiver, J.W. and Liu, M.A. (1997). DNA vaccines. Annu Rev Immunol 15, 617-48.
8.	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.
9.	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.
10.	Svab, Z., Hajdukievitz, P. and Maliga, P. (1990). Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87, 8526-8530.
11.	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.
12.	Sanford, J.C., Smith, F.D. and Russell, J.A. (1993). Optimization of the biolistic process for different bioloical applications. Methods of Enzymology 217, 483-509.
13.	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