In genetic engineering, a gene gun or a biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. The payload is an elemental particle of a heavy metal coated with the molecule of interest (typically plasmid DNA). This technique is often 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 and mitochondria.
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 the device was used to deliver particles coated with a marker gene which would relay a signal if proper insertion of the DNA transcript occurred. Genetic transformation was the demonstrated upon observed expression of the marker gene within onion cells.
The earliest custom manufactured gene guns (fabricated by Nelson Allen) used a 22 caliber nail gun cartridge to propel a polyethylene cylinder (bullet) down a 22 caliber Douglas barrel. A droplet of the tungsten powder coated with genetic material was placed onto the bullet and shot down into a Petri dish below. The bullet welded to the disk below the Petri plate, and the genetic material blasted into the sample with a doughnut effect involving devastation in the middle of the sample with a ring of good transformation around the periphery. The gun was connected to a vacuum pump and was placed under a vacuum while firing. The early design was put into limited production by a Rumsey-Loomis (a local machine shop then at Mecklenburg Road in Ithaca, NY, USA).
Biorad contracted with Dupont to manufacture and distribute an updated device with improvements including the use of helium as a non-explosive propellant and a multi-disk collision delivery mechanism to minimize damage to sample tissues. Other heavy metals such as gold and silver are also used to deliver genetic material with gold being favored due to lower cytotoxicity in comparison to tungsten projectile carriers.
Biolistic construct design
A construct is a gene or series of genes with required regulatory elements that is designed for insertion into foreign genome. 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 and 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 cells which did not successfully receive and integrate the construct into their DNA. These genes may each have their own promoter and terminator, or they can be grouped to produce multiple gene products from one transcript. The construct may be flanked by border sequences which are homologous to particular locations within the target genome, allowing the construct to target and integrate itself in a particular designated region.
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 mostly used with 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 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 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.
Biolistics 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. The delivery and integration of multiple templates of the DNA construct is a distinct possibility, resulting in potential variable expression levels and copy numbers of the inserted gene. 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, they cannot be targeted to specific locations within the genome, unless the transgene is co-delivered with genome editing reagents.
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- John O'Brien presents...Gene Gun Barrels for more information about biolistics