In 1972 for the first time, Herbert boyer and Stanely Cohen introduced a cathartic technique of genetic engineering, The Gene gun, which involved a metamorphosis of genetic material within organisms. The reconstruction of the genome accomplished through a series of advancements in the field of genetic engineering that led the scientists to modify the genome by their own choice. A headway includes the innovation of DNA ligases, restriction enzymes, the ability to construct plasmid and PCR.
In 1974 Rudolf Jaenisch, one of the pioneering scientists in the field of genetic engineering, for the first time was successful in modifying the genome of mice. The technology hit its peak level, and it became commercialized in 1976 with the introduction of genetically modified bacteria that produced somatostatin. In 1978 scientists designed bacteria that produced life dependent pills for diabetic patients i-e insulin. It was in 1983 when for the first time scientists introduced an antibiotic-resistant gene in the tobacco plant. Scientists then used this tool to remove the unfavorable genes and to add genes that ensure survival for the organism.
FORTUNATE BIOLOGICAL TOOL
Gene gun is a special tool/procedure for plant transformation. Gene gun carries the DNA from outside to the inside of the cell. In short, the gene gun helps in transgenesis. Apart from the transformation of exogenous DNA, but it is also used in the transformation of the nucleus and organelles like plasmid, etc.
Gene gun was originally designed by john c Sanford and his colleagues at Cornell University between 1983 and 1986. The first time, the golden gun (gene gun, Golden because gold particles were utilized) was applied on onion cells because of its large size cell composition.
Gene gun design
The following components are the key features of a gene gun.
- Gun cartridge with a size of .22 calibers.
- Bullet portion which fits exactly in the gun cartridge. The bullet is polyethylene in nature. Furthermore, the bullet portion contains tungsten powder and genetic material.
- Lexan (polycarbonate resin thermoplastic) is use as the stopping disk. Below the stopping disk, Petri plates are present, which contains the target specimen.
- Helium propellant as a non-exclusive propellant.
- Gold and silver are used as a heavy metal instead of tungsten because of its fine symmetry and are non-toxic in nature to the cell.
How it works
It involved the following steps
- Within the gene gun apparatus, the specimen is position in a way so that it is ready to fire.
- The core chamber of the gene gun fills with helium and in turn build pressure on the rupture disk point
- The pressure in the core chamber constrained the rupture point to break, eventually the helium burst. The energy released during the bombardment used to kick the DNA gold-coated macro carrier into the stopping screen
- After hold out to the stopping screen, the carrier DNA finds its way to the targeted cell.
Gene gun was originally for plant specimens, but we can also use it for animal and human specimens.
In plants, The specimen for transformation is callus (mass of undifferentiated cells ). The specimen may be an immature embryo, growing in a petri dish under a controlled environment. The transformation of the specimen occurs with the help of the gene gun, via target DNA. The DNA which enters into the target cell has two possibilities, either it expresses itself through transcription or it enters into the chromosome of the target cell (stable transformation). The carrier DNA contains a selectable marker, based on which we predict whether the DNA colonized the cell or not. For example, if the carrier DNA contains a gene for antibiotic resistance.
After transformation, we treat the selected culture with a particular antibiotic for which a carrier DNA contain the resistant gene. If the gene is present then the cell survives and if it is not present then it vanishes. Plant hormones i-e auxin and gibberellin are then added to the culture containing carrier DNA under laboratory-controlled conditions. After encountering the plant hormones, the undifferentiated mass of the cell became organized and develop into a tissue. As a result, the new plant develops while containing the carrier DNA. Which is now transferable from one generation to another.
Humans and other Animals
Gene gun plays a pivoted role in DNA vaccination. With a gene gun, we insert plasmid (extrachromosomal ring) into rat neurons, specifically in DRG (dorsal root ganglion) neurons. Sometimes the plasmid insert into the targeted cell is used as a pharmaceutical precursor for the study of devastating neurodegenerative diseases such as Alzheimer’s disease.
Apart from this, we can insert a gene of interest into the extrachromosomal DNA or plasmid. The insertion is performed for the identification of a specific gene with the help of a radioactive label fluorescent dye mechanism.in addition to this, we can also extract the group of culture from the media by this tool by using the insertion of selectable marker genes that possess the specific ability of defense against certain anti agents such as antibiotics. For example, we applied certain specific antibiotics to the whole culture. Those culture of living organism survives which possesses gene to combat with foreign particles.
An explanation of how gene gun works
Transformation of DNA particle with the help of a gene gun is more accurate and stable than other previous techniques such as agrobacterium mediated transformation. Because in the agrobacterium mediated transformation technique, the insertion of targeted particles into the vector is difficult. Biolistics technique produced successful resistant crops. Bt maize is one of the exemplary products of biolistics. Moreover, with this technique, a well-skilled technician can produce two transgenes with a single fire from a gene gun. Even biolistic allow us to work on tissue in situ.
The technique is so far effective in this regard but still, have some kind of defects. One of the serious issue with this technique is that the DNA transform randomly into the targeted cell. When the carrier DNA enters into the cell it possesses a probability of inserting into plasmid, mitochondria, and nucleus. Here the problem is with the plasmid and mitochondrial genome which affect the final result. Another issue is that the targeted DNA may insert multiple times which causes overexpression and hence affect the final result.