Gene gun

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PDS-1000/He Particle Delivery System Genegun.jpg
PDS-1000/He Particle Delivery System

In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein 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". [1] [2]

Contents

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 and mitochondria. [3]

A gene gun is used for delivery of exogenous DNA to cells. This method is known as 'biolistics'. Gene guns can be used effectively on most cells but are mainly used on plant cells. The gene gun apparatus is ready to fire. Helium fills the chamber and pressure builds against the rupture disk. The pressure eventually reaches the point where the rupture disk breaks, and the resulting burst of helium propels the DNA/gold-coated macrocarrier ('Plastic Disk') into the stopping screen. When the macrocarrier hits the stopping screen, the DNA-coated gold particles are propelled through the screen and into the target cells. Gene Gun.svg
A gene gun is used for delivery of exogenous DNA to cells. This method is known as 'biolistics'. Gene guns can be used effectively on most cells but are mainly used on plant cells.
  1. The gene gun apparatus is ready to fire.
  2. Helium fills the chamber and pressure builds against the rupture disk.
  3. The pressure eventually reaches the point where the rupture disk breaks, and the resulting burst of helium propels the DNA/gold-coated macrocarrier ('Plastic Disk') into the stopping screen.
  4. When the macrocarrier hits the stopping screen, the DNA-coated gold particles are propelled through the screen and into the target cells.

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 [4] [5] [6] along with 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. [7] 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 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).

Biolistics, Inc sold Dupont the rights 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. [8]

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. [9] [ page needed ] 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 sequence, the gene of interest, and a reporter gene.

Promoter
Promoters control the location and magnitude of gene expression and function as “the steering wheel and gas pedal” of a gene. [9] [ page needed ] 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 is an example of a commonly used promoter that results in robust constitutive gene expression within plants. [10]
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 . 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. [11] [ failed verification ]
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, hygromycin B, or glyphosate [9] [ page needed ]. [12] [13] [14]
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. [15] 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 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. [16]

Gene gun bombardment has also been used to transform Caenorhabditis elegans , as an alternative to microinjection. [17]

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. Notably, Bt maize is a product of biolistics. [9] [ page needed ] 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. [9] [ page needed ] [18] In addition, there are no reports of a chloroplast silencing a transgene inserted with a gene gun. [19] Additionally, with only one firing of a gene gun, a skilled technician can generate two transformed organisms in certain species. [18] 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. [20]

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. [9] [ page needed ] 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. [9] [ page needed ] 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, [9] [ page needed ] unless the transgene is co-delivered with genome editing reagents.

Related Research Articles

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Reporter gene</span> Technique in molecular biology

In molecular biology, a reporter gene 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 characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

<span class="mw-page-title-main">Yeast artificial chromosome</span> Genetically engineered chromosome derived from the DNA of yeast

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

<span class="mw-page-title-main">Transfer DNA</span> Type of DNA in bacterial genomes

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

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 phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

<span class="mw-page-title-main">Transplastomic plant</span>

A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.

A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a T-DNA binary vector and a virhelper plasmid. The two plasmids are used together to produce genetically modified plants. They are artificial vectors that have been derived from the naturally occurring Ti plasmid found in bacterial species of the genus Agrobacterium, such as A. tumefaciens. The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts.

<span class="mw-page-title-main">Insert (molecular biology)</span>

In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

References

  1. ↑ O'Brien, John A.; Lummis, Sarah CR (2011). "Nano-biolistics: A method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles". BMC Biotechnology. 11: 66. doi: 10.1186/1472-6750-11-66 . PMC   3144454 . PMID   21663596.
  2. ↑ Carter, Matt; Shieh, Jennifer (6 March 2015). "Chapter 11 - Gene Delivery Strategies". Academic Press (Second ed.). ISBN   978-0-12-800511-8.
  3. ↑ Sanford, John C. (1990). "Biolistic plant transformation". Physiologia Plantarum. 79 (1): 206–209. doi:10.1111/j.1399-3054.1990.tb05888.x. ISSN   1399-3054.
  4. ↑ Segelken, Roger (14 May 1987). "Biologist invent gun for shooting cells with DNA" (PDF). Cornell Chronicle. p. 3. Archived from the original (PDF) on 29 October 2013. Retrieved 5 June 2014.
  5. ↑ 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.
  6. ↑ 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 (6117): 70–73. Bibcode:1987Natur.327...70K. doi:10.1038/327070a0. S2CID   4265777.
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  8. ↑ Russell, Julie A.; Roy, Mihir K.; Sanford, John C. (1992-03-01). "Physical Trauma and Tungsten Toxicity Reduce the Efficiency of Biolistic Transformation". Plant Physiology. 98 (3): 1050–1056. doi:10.1104/pp.98.3.1050. ISSN   0032-0889. PMC   1080307 . PMID   16668726.
  9. 1 2 3 4 5 6 7 8 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.
  10. ↑ Benfey, P. N.; Chua, N.-H. (1990-11-16). "The Cauliflower Mosaic Virus 35S Promoter: Combinatorial Regulation of Transcription in Plants". Science. 250 (4983): 959–966. Bibcode:1990Sci...250..959B. doi:10.1126/science.250.4983.959. ISSN   0036-8075. PMID   17746920. S2CID   35471862.
  11. ↑ "nopaline synthase terminator: Topics by Science.gov". www.science.gov. Retrieved 2019-11-20.
  12. ↑ Norris, M. H.; Kang, Y.; Lu, D.; Wilcox, B. A.; Hoang, T. T. (2009-07-31). "Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei". Applied and Environmental Microbiology. 75 (19): 6062–6075. Bibcode:2009ApEnM..75.6062N. doi:10.1128/aem.00820-09. ISSN   0099-2240. PMC   2753064 . PMID   19648360.
  13. ↑ Blochlinger, K; Diggelmann, H (December 1984). "Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells". Molecular and Cellular Biology. 4 (12): 2929–2931. doi:10.1128/mcb.4.12.2929. ISSN   0270-7306. PMC   369308 . PMID   6098829.
  14. ↑ Carrer, Helaine; Hockenberry, Tish Noel; Svab, Zora; Maliga, Pal (October 1993). "Kanamycin resistance as a selectable marker for plastid transformation in tobacco". MGG Molecular & General Genetics. 241–241 (1–2): 49–56. doi:10.1007/bf00280200. ISSN   0026-8925. PMID   8232211. S2CID   2291268.
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  16. ↑ Gan, Wen-Biao; Grutzendler, Jaime; Wong, Wai Thong; Wong, Rachel OL; 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. S2CID   16962732.
  17. ↑ Praitis, Vida (2006). "Creation of Transgenic Lines Using Microparticle Bombardment Methods". C. Elegans. Methods in Molecular Biology. Vol. 351. pp. 93–108. doi:10.1385/1-59745-151-7:93. ISBN   978-1-59745-151-2. PMID   16988428.
  18. 1 2 Sanford, John (April 28, 2006). "Biolistic plant transformation". Physiologia Plantarum. 79 (1): 206–209. doi:10.1111/j.1399-3054.1990.tb05888.x.
  19. ↑ Kikkert, Julie; Vidal, Jose; Reisch, Bruce (2005). "Stable Transformation of Plant Cells by Particle Bombardment/Biolistics". Transgenic Plants. Methods in Molecular Biology. Vol. 286. pp. 61–78. doi:10.1385/1-59259-827-7:061. ISBN   978-1-59259-827-4. PMID   15310913. S2CID   44395352.
  20. ↑ Hayward, M.D.; Bosemark, N.O.; Romagosa, T. (2012). Plant Breeding: Principles and Prospects. Springer Science & Business Media. p. 131. ISBN   9789401115247.

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