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Biolistics is the technique of bombarding a biomolecule especially nucleic acid in to a living cell using the particle bombardment technique.Using the sofisticated gene gun, it is easy to insert a piece of nucleic acid into the cell to express its character.This method is sucessfully carried out in many monocotyledonous and dicotyledonous plants.
Biolistics or Particle bombardment is a relatively simple laboratory technique.Tungsten or gold beads smaller than the plant cells containing the gene of interest are fired into the cell through a stopping screen accelerated ( with very high velocity of 1,400 ft per second) by helium.As the particle enters into the cell, DNA will be released.The technique is also known as Microprojectiles or Particle gun or Particle bombardment technique. After bombardment, the transformed tissue is selected using marker genes like one coding for antibiotics resistance.From the transformed tissue, whole plants can be generated with a copy of the transgene in every cell.
These are particles used for bombarding the gene of interest. The particles may be tungsten or gold beads having a diameter of 1pm. The gene to be trasferred lies in side the beads and the whole structure is called as bullet.The target cells for these bullets can be living plant cells such as pollen, meristems, cultured cells etc.
Firing the bullets
For firing the bullets, high velocity acceleration technique is used. The acceleration of the bullets is achieved by either Explosive Charge method ( Corrosive explosion) or by Shock wave produced through high voltage electric discharge. Usually high velocity of 1400 ft/second is used.
It is the system to fire the Microprojectiles into the cells. The Gene gun is also known as Biolistics Particle Delivery system.This system is developed originally for plant transformation. The gene gun method is also referred to as Bioballistics method and can be used to transfer any cell organalle including plastids. The gene gun was orginally a Crossman air pistol used for firing tugsten particles.This gun was intially used to deliver marker genes into onion cells. The earlier gene gun used a 22 caliber nail gun cartridge to fire an extruded polyethylene bullet.The technique used a droplet of tugsten powder mixed with the DNA . This mixture was placed on the bullet for firing and the bullets are fired to a stopping dick with a petri dish below.The bullet stick on the disc while the DNA released into the sample in the petri dish. Now a days, improved guns are used with Helium propellant and Mask disc collision delivery mechanism.Besides tugsten, other heavy metals such as gold, silver etc are also used as bullets.Gold is considered as good due to its better uniformity than tungsten. The tugsten can be toxic to cells sometimes.
The gene gun technique is also used in human beings and animals to deliver DNA vaccins.Genegun technique is used to deliver plasmids into the rat neurons to study the effect of diseases like Alzheimer’s disease. The genegun technique is also used in edible vaccine. The target of the gene gun is a callus of undifferentiated plant cells cultured in the culture medium. When the bullets, hit on the culture, the medium and the cells disrupts.Some of the cells take the bullets during hitting and the DNA migrate into the cell and integrate with the chromosome.Using modern biochemical methods, it is possible to recollect the integrated cells.Tandem Selectable gene and Northern blotting methods are used for this purpose. The selected transformed cells are then treated with plant hormones like Auxins and Gibberellins.Each transformed cell multiply and differentiate into a plant with genetic transformation.This totipotent Shot Cell has the capability to differentiate into a full fledged plant with the genetic transformation.Gene gun is also used to deliver vital dyes into cells to study the internal featurs of the cells.Using the biolistic method, various breeds of tobacco, rice, maize, wheat etc have been produced.
A sample protocol of gene transfer.
Target cells are Rice cells callus .DNA sample used is 6 to 8 kb.Bullet size is 1.5 microgram
Microcarrier used may be Gold or tugsten bullet of 1 micron size.Bombradment quantity of particle is 1 mg.Pressure applied is 1100 psi. Target distance of bombardment is 4 cm / twice at 8 cm.
Culturing involves induction on NB6 medium with 2,4-D ( 2mg per litre). This is followed by transferring the callus into NBO a mixture of NB6, 2,4-D, 0.2 M sorbitol and 0.2M mannitol.This is followed by transformation of the cells with PDS-1000 / He. The cells are then transferred to NH40 selection ( NB6, 40mg / litre Hygromycin).
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