GENE GUN AND DESIGN
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 induced into cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics. 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.
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 sequence, the gene of interest, and a reporter gene.
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 is an example of a commonly used promoter that results in robust constitutive gene expression within plants.
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.
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
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 preform specialized functions alongside the main gene of interest.
Gene guns are mostly used with plant cells. However, there is much potential use in humans and other animals as well.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.Gene gun bombardment has also been used to transform Caenorhabditis elegans, as an alternative to microinjection