A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for delivering exogenous DNA ( transgenes) to cells. The payload is an elemental particle of a heavy metal coated with DNA (typically plasmid DNA). This technique is often simply referred to as biolistics. This device is able to transform almost any type of cell, including plants, and is not limited to transformation of the nucleus; it can also transform organelles, including plastids.
Gene gun designThe 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 material 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 designA 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 enable 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.
ApplicationGene guns are so far mostly used with plant cells. However, there is much potential use in humans and other animals as well.
PlantsThe target of a gene gun is often a callus of undifferentiated plant cells or a group of immature embryos growing on gel medium in a Petri dish. After the DNA-coated gold particles have been delivered to the cells, the DNA is used as a template for transcription (transient expression) and sometimes it integrates into a plant chromosome ('stable' transformation) If the delivered DNA construct contains a selectable marker, then stably transformed cells can be selected and cultured using tissue culture methods. For example, if the delivered DNA construct contains a gene that confers resistance to an antibiotic or herbicide, then stably transformed cells may be selected by including that antibiotic or herbicide in the tissue culture media. Transformed cells 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 transformed cell may have new traits that are heritable. The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert DNA into plant cells. See transformation for different methods of transformation in different species.
Humans and other animalsGene 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. Gene gun bombardment has also been used to transform Caenorhabditis elegans, as an alternative to microinjection.
AdvantagesBiolistics 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.
LimitationsBiolistics introduces DNA randomly into the target cells. Thus the DNA 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 material 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, unless the transgene is co-delivered with genome editing reagents.
- John O'Brien presents...Gene Gun Barrels for more information about biolistics