Gene gun
From Wikipedia, the free encyclopedia
The gene gun is a device for injecting cells with genetic information, originally designed for plant transformation. The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as biolistics. Another instrument that uses biolistics technology is the PDS-1000/He particle delivery system (pictured).
This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.
[edit] Design
The gene gun was originally a Crossman air pistol modified to fire dense tungsten particles and was first used on an onion to achieve genetic transformation with a phosphorescent marker gene. The earliest custom manufactured geneguns (Fabricated by Nelson Allen) used a 22 caliber nailgun 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 information 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 Mechlenburg Rd in Ithaca, NY). Later the design was refined by removing the "surge tank" and changing to nonexplosive propellants. DuPont added a plastic extrusion to the exterior to "prettify" 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.
The primary inventors of the Gene gun are Horticultural scientist John C. Sanford who took his need for inserting genes into cells to Edward Wolf (Then Director of Cornells Submicron Facility now Nanofabrication facility). Wolf (an Electrical Engineer) familiar with making and using small structures came bought the Crossman air pistol and performed the first genegun experiments with it in his basement (Sanford would come to his house with the genetic material and then take the transformed cells back to his lab). Horticultural scientist Theodore Klein at Cornell University worked closely with John Sanford on experiments using and proving the genegun. They had support from Nelson Allen of the Cornell Nanofabrication Facilities Machine shop (a coinventor) who had an instrumental role in changing the genegun from the air pistol prototype to a working scientific device. The rights to commercial use of the gene gun were sold by Wolf, Sanford and Cornell University to DuPont in 1990.
[edit] Application
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the tungsten particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated tungsten particle, whose DNA eventually migrates to and integrates into a plant chromosome.
Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots.
Selected single cells from the callus 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 shot cell may have new genetic (heritable) traits.
Using the gene gun may be contrasted with using Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species.
Gene guns have also been used to deliver DNA vaccines to experimental animals.
The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG neurons, is also used as a pharmalogical precursor in studying the effects of neurodegenerative diseases such as Alzheimer's Disease.
