In
genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
, a gene gun or biolistic particle delivery system is a device used to deliver exogenous
DNA (
transgene
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
s),
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
, or
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
to cells. By coating particles of a
heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be introduced into desired cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics, short for "biological ballistics".
This device is able to transform almost any type of cell and is not limited to the transformation of the nucleus; it can also transform organelles, including
plastids
The plastid (Greek: πλαστός; plastós: formed, molded – plural plastids) is a membrane-bound organelle found in the cells of plants, algae, and some other eukaryotic organisms. They are considered to be intracellular endosymbiotic cyanobac ...
and
mitochondria
A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
.
Gene gun design
The gene gun was originally a
Crosman
Crosman Corporation is an American designer, manufacturer and supplier of shooting sport products, with a long-standing presence in airgun design and a tradition of producing pellet and BB guns. Crosman is also a producer of many varieties of a ...
air pistol
The atmosphere of Earth is the layer of gases, known collectively as air, retained by Earth's gravity that surrounds the planet and forms its planetary atmosphere. The atmosphere of Earth protects life on Earth by creating pressure allowing for ...
modified to fire dense
tungsten
Tungsten, or wolfram, is a chemical element with the symbol W and atomic number 74. Tungsten is a rare metal found naturally on Earth almost exclusively as compounds with other elements. It was identified as a new element in 1781 and first isolat ...
particles. It was invented by
John C Sanford, Ed Wolf, and Nelson Allen at
Cornell University
Cornell University is a private statutory land-grant research university based in Ithaca, New York. It is a member of the Ivy League. Founded in 1865 by Ezra Cornell and Andrew Dickson White, Cornell was founded with the intention to teach an ...
along with Ted Klein of
DuPont
DuPont de Nemours, Inc., commonly shortened to DuPont, is an American multinational chemical company first formed in 1802 by French-American chemist and industrialist Éleuthère Irénée du Pont de Nemours. The company played a major role in ...
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 In biology, a marker gene may have several meanings. In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. In particular, there are tw ...
which would relay a signal if proper insertion of the DNA transcript occurred.
Genetic transformation was 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
A nail gun, nailgun or nailer is a form of hammer used to drive nails into wood or other materials. It is usually driven by compressed air (pneumatic), electromagnetism, highly flammable gases such as butane or propane, or, for powder-actuate ...
cartridge to propel a
polyethylene
Polyethylene or polythene (abbreviated PE; IUPAC name polyethene or poly(methylene)) is the most commonly produced plastic. It is a polymer, primarily used for packaging ( plastic bags, plastic films, geomembranes and containers including bo ...
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
A Petri dish (alternatively known as a Petri plate or cell-culture dish) is a shallow transparent lidded dish that biologists use to hold growth medium in which cells can be cultured,R. C. Dubey (2014): ''A Textbook Of Biotechnology For Class- ...
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).
Biolistics, Inc sold Dupont the rights to manufacture and distribute an updated device with improvements including the use of
helium
Helium (from el, ἥλιος, helios, lit=sun) is a chemical element with the symbol He and atomic number 2. It is a colorless, odorless, tasteless, non-toxic, inert, monatomic gas and the first in the noble gas group in the periodic table. ...
as a non-explosive propellant and a multi-disk collision delivery mechanism to minimize damage to sample tissues. Other heavy metals such as
gold
Gold is a chemical element with the symbol Au (from la, aurum) and atomic number 79. This makes it one of the higher atomic number elements that occur naturally. It is a bright, slightly orange-yellow, dense, soft, malleable, and ductile met ...
and
silver
Silver is a chemical element with the Symbol (chemistry), symbol Ag (from the Latin ', derived from the Proto-Indo-European wikt:Reconstruction:Proto-Indo-European/h₂erǵ-, ''h₂erǵ'': "shiny" or "white") and atomic number 47. A soft, whi ...
are also used to deliver genetic material with gold being favored due to lower cytotoxicity in comparison to tungsten projectile carriers.
Biolistic construct design
Biolistic transformation involves the integration of a functional fragment of DNA—known as a DNA construct—into target cells. A gene construct is a DNA cassette containing all required regulatory elements for proper expression within the target organism.
While gene constructs may vary in their design depending on the desired outcome of the transformation procedure, all constructs typically contain a combination a
promoter sequence, a
terminator
Terminator may refer to:
Science and technology
Genetics
* Terminator (genetics), the end of a gene for transcription
* Terminator technology, proposed methods for restricting the use of genetically modified plants by causing second generation s ...
sequence, the gene of interest, and a
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
.
Promoter:
Promoters control the location and magnitude of gene expression and function as “the steering wheel and gas pedal” of a gene.
Promoters precede the gene of interest in the DNA construct and can be changed through laboratory design to fine-tune transgene expression. The 35S promoter from
Cauliflower mosaic virus
Cauliflower mosaic virus (CaMV) is a member of the genus ''Caulimovirus'', one of the six genera in the family ''Caulimoviridae'', which are pararetroviruses that infect plants. Pararetroviruses replicate through reverse transcription just like ...
is an example of a commonly used promoter that results in robust constitutive gene expression within plants.
Terminator:
Terminator sequences are required for proper gene expression and are placed after the coding region of the gene of interest within the DNA construct. A common terminator for biolistic transformation is the NOS terminator derived from
Agrobacterium tumefaciens
''Agrobacterium radiobacter'' (more commonly known as ''Agrobacterium tumefaciens'') is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Sympto ...
. Due to the high frequency of use of this terminator in genetically engineered plants, strategies have been developed to detect its presence within the food supply to monitor for unauthorized GE crops.
Reporter gene:
A gene encoding a selectable marker is a common element within DNA constructs and is used to select for properly transformed cells. The selectable marker chosen will depend on the species being transformed, but it will typically be a gene granting cells a detoxification capacity for certain herbicides or antibiotics such as
kanamycin
Kanamycin A, often referred to simply as kanamycin, is an antibiotic used to treat severe bacterial infections and tuberculosis. It is not a first line treatment. It is used by mouth, injection into a vein, or injection into a muscle. Kanamycin ...
,
hygromycin B
Hygromycin B is an antibiotic produced by the bacterium '' Streptomyces hygroscopicus''. It is an aminoglycoside that kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis.
History
Hygromycin B was originally develo ...
, or
glyphosate
Glyphosate (IUPAC name: ''N''-(phosphonomethyl)glycine) is a broad-spectrum Herbicide, systemic herbicide and Crop desiccation, crop desiccant. It is an organophosphorus compound, specifically a phosphonate, which acts by inhibiting the plan ...
.
Additional elements:
Optional components of a DNA construct include elements such as
cre-lox sequences that allow for controlled removal of the construct from the target genome. Such elements are chosen by the construct developer to perform specialized functions alongside the main gene of interest.
Application
Gene guns are mostly used with plant cells. However, there is much potential use in humans and other animals as well.
Plants
The target of a gene gun is often a
callus
A callus is an area of thickened and sometimes hardened skin that forms as a response to repeated friction, pressure, or other irritation. Since repeated contact is required, calluses are most often found on the feet and hands, but they may o ...
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
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
('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
Auxins (plural of auxin ) are a class of plant hormones (or plant-growth regulators) with some morphogen-like characteristics. Auxins play a cardinal role in coordination of many growth and behavioral processes in plant life cycles and are essenti ...
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 Pluripotency: These are the cells that can generate into any of the three Germ layers which imply Endodermal, Mesodermal, and Ectodermal cells except tissues like the placenta.
According to Latin terms, Pluripotentia means the ability for many thin ...
. 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
''Agrobacterium radiobacter'' (more commonly known as ''Agrobacterium tumefaciens'') is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Sympto ...
'' and its
Ti plasmid
A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of ''Agrobacterium'', including ''A. tumefaciens, ''A. rhizogenes'', ''A. rubi'' and ''A. vitis''.
Evolutionarily, the Ti plasmid is part of a family of plasmids carried b ...
to insert DNA into plant cells. See
transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Trans ...
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
Alzheimer's disease (AD) is a neurodegeneration, neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in short-term me ...
.
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
Transform may refer to:
Arts and entertainment
* Transform (scratch), a type of scratch used by turntablists
* ''Transform'' (Alva Noto album), 2001
* ''Transform'' (Howard Jones album) or the title song, 2019
* ''Transform'' (Powerman 5000 album ...
''
Caenorhabditis elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (ro ...
'', as an alternative to
microinjection
Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical pro ...
.
Advantages
Biolistics has proven to be a versatile method of genetic modification and it is generally preferred to engineer transformation-resistant crops, such as
cereals
A cereal is any grass cultivated for the edible components of its grain (botanically, a type of fruit called a caryopsis), composed of the endosperm, germ, and bran. Cereal grain crops are grown in greater quantities and provide more food en ...
. 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 in certain species.
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.
Limitations
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
Illegitimate recombination, or nonhomologous recombination, is the process by which two unrelated double stranded segments of DNA are joined. This insertion of genetic material which is not meant to be adjacent tends to lead to genes being broken ...
—a process by which the transgene is integrated into the genome without similar genetic sequences—and not
homologous recombination
Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in cellular organisms but may ...
, they cannot be targeted to specific locations within the genome,
unless the transgene is co-delivered with
genome editing
Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts ...
reagents.
References
Further reading
*
External links
John O'Brien presents...Gene Gun Barrelsfor more information about biolistics
{{DEFAULTSORT:Gene Gun
Molecular biology
Molecular genetics
Laboratory techniques
Gene delivery
1983 introductions
Nanotechnology