A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information; the inserted genetic material are termed transgenes. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.
This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.
Gene gun design
The gene gun was originally a Crosman air pistol modified to fire dense tungsten particles. It was invented by John C Sanford, Ed Wolf and Nelson Allen at Cornell University, and Ted Klein of DuPont, between 1983 and 1986. The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with a marker gene. Genetic transformation was then proven when the onion tissue expressed the gene.
The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 caliber nail gun cartridge to propel an extruded polyethylene cylinder (bullet) down a 22 cal. Douglas barrel. A droplet of the tungsten powder and genetic material was placed on the bullet and shot down the barrel at a lexan "stopping" disk with a petri dish below. The bullet welded to the disk and the genetic information blasted into the sample in the dish with a doughnut effect (devastation in the middle, a ring of good transformation and little around the edge). The gun was connected to a vacuum pump and was under vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Rd in Ithaca, NY, USA). Later the design was refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to visually improve the machine for mass production to the scientific community. Biorad contracted with Dupont to manufacture and distribute the device. Improvements include the use of helium propellant and a multi-disk-collision delivery mechanism. Other heavy metals such as gold and silver are also used. Gold may be favored because it has better uniformity than tungsten and tungsten can be toxic to cells, but its use may be limited due to availability and cost.
Biolistic construct design
A construct is a piece of DNA inserted into the target's genome, including parts that are intended to be removed later. All biolistic transformations require a construct to proceed and while there is great variation among biolistic constructs, they can be broadly sorted into two categories: those which are designed to transform eukaryotic nuclei, and those designed to transform prokaryotic-type genomes such as mitochondria, plasmids or plastids.
Those meant to transform prokaryotic genomes generally have the gene or genes of interest, at least one promoter and terminator sequence, and a reporter gene; which is a gene used to ease detection or removal of those cells which didn't integrate the construct into their DNA. These genes may each have their own promoter and terminator, or be grouped to produce multiple gene products from one transcript, in which case binding sites for translational machinery should be placed between each to ensure maximum translational efficiency. In any case the entire construct is flanked by regions called border sequences which are similar in sequence to locations within the genome, this allows the construct to target itself to a specific point in the existing genome.
Constructs meant for integration into a eukaryotic nucleus follow a similar pattern except that: the construct contains no border sequences because the sequence rearrangement that prokaryotic constructs rely on rarely occurs in eukaryotes; and each gene contained within the construct must be expressed by its own copy of a promoter and terminator sequence.
Though the above designs are generally followed, there are exceptions. For example, the construct might include a Cre-Lox system to selectively remove inserted genes; or a prokaryotic construct may insert itself downstream of a promoter, allowing the inserted genes to be governed by a promoter already in place and eliminating the need for one to be included in the construct.
Gene guns are so far mostly applied for plant cells. However, there is much potential use in humans and other animals as well.
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a Petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into a plant chromosome.
Cells from the entire Petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots.
Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits.
The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species.
Humans and other animals
Gene guns have also been used to deliver DNA vaccines.
The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG neurons, is also used as a pharmacological precursor in studying the effects of neurodegenerative diseases such as Alzheimer's disease.
The gene gun has become a common tool for labeling subsets of cells in cultured tissue. In addition to being able to transfect cells with DNA plasmids coding for fluorescent proteins, the gene gun can be adapted to deliver a wide variety of vital dyes to cells.
Biolistics has proven to be a versatile method of genetic modification and it is generally preferred to engineer transformation-resistant crops, such as cereals. Notably, Bt maize is a product of biolistics. Plastid transformation has also seen great success with particle bombardment when compared to other current techniques, such as Agrobacterium mediated transformation, which have difficulty targeting the vector to and stably expressing in the chloroplast. In addition, there are no reports of a chloroplast silencing a transgene inserted with a gene gun. Additionally, with only one firing of a gene gun, a skilled technician can generate two transformed organisms. This technology has even allowed for modification of specific tissues in situ, although this is likely to damage large numbers of cells and transform only some, rather than all, cells of the tissue.
However, biolistics introduces the construct randomly into the target cells. Thus the altered DNA sequences may be transformed into whatever genomes are present in the cell, be they nuclear, mitochondrial, plasmid or any others, in any combination, though proper construct design may mitigate this. Another issue is that the gene inserted may be overexpressed when the construct is inserted multiple times in either the same or different locations of the genome. This is due to the ability of the constructs to give and take genetic information from other constructs, causing some to carry no transgene and others to carry multiple copies; the number of copies inserted depends on both how many copies of the transgene an inserted construct has, and how many were inserted. Also, because eukaryotic constructs rely on illegitimate recombination, a process by which the transgene is integrated into the genome without similar genetic sequences, and not homologous recombination, which inserts at similar sequences, they cannot be targeted to specific locations within the genome.
- Segelken, Roger (14 May 1987). "Biologist invent gun for shooting cells with DNA" (PDF). Cornell Chronicle. p. 3. Retrieved 5 June 2014.
- Sanford, J.C.; Klein, T.M.; Wolf, E.D.; Allen, N. (1987). "Delivery of substances into cells and tissues using a particle bombardment process". Particulate Science and Technology. 5 (1): 27–37. doi:10.1080/02726358708904533.
- Klein, T.M.; Wolf, E.D.; Wu, R.; Sanford, J.C. (May 1987). "High-velocity microprojectiles for delivering nucleic acids into living cells". Nature. 327: 70–73. doi:10.1038/327070a0.
- Segelken, Roger. "The Gene Shotgun". Cornell University College of Agriculture and Life Sciences. Archived from the original on 26 April 2010. Retrieved 5 June 2014.
- Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology: the genetic manipulation of plants (2 ed.). Oxford, New York, USA: Oxford University Press Inc. ISBN 978-0-19-928261-6.
- Gan, Wen-Biao; Grutzendler, Jaime; Wong, Wai Thong; Wong, Rachel O.L; Lichtman, Jeff W (2000). "Multicolor "DiOlistic" Labeling of the Nervous System Using Lipophilic Dye Combinations". Neuron. 27 (2): 219–25. doi:10.1016/S0896-6273(00)00031-3. PMID 10985343.
- Praitis, Vida (2006). "Creation of Transgenic Lines Using Microparticle Bombardment Methods". 351: 93–108. doi:10.1385/1-59745-151-7:93. ISBN 1-59745-151-7.
- Sanford, John (April 28, 2006). "Biolistic plant transformation". Physiologia Plantarum. 79 (1): 206–209. doi:10.1111/j.1399-3054.1990.tb05888.x. Retrieved 16 October 2015.
- Kikkert, Julie; Vidal, Jose; Reisch, Bruce (2005). "Stable transformation of plant cells by particle bombardment/bioistics". Methods of Molecular Biology. 286: 61–78. doi:10.1385/1-59259-827-7:061. PMID 15310913. Retrieved 16 October 2015.
- Hayward, M.D.; Bosemark, N.O.; Romagosa, T. (2012). Plant Breeding: Principles and Prospects. Springer Science & Business Media. p. 131. ISBN 9789401115247.
- O'Brien, J; Holt, M; Whiteside, G; Lummis, SC; Hastings, MH (2001). "Modifications to the hand-held Gene Gun: improvements for in vitro Biolistic transfection of organotypic neuronal tissue". Journal of Neuroscience Methods. 112 (1): 57–64. doi:10.1016/S0165-0270(01)00457-5. PMID 11640958.
- John O'Brien presents...Gene Gun Barrels for more information about biolistics