Biolistics

A NEW WAY OF INJECTING GENETIC INFORMATION

1. biolistics A NEW WAY OF INJECTING GENETIC INFORMATION……………

2. CONTENTS……………. What is biolistics???????? Gene gun design Biolistics construct design Applications Plants Human and other animals Advantages limitations

3. Biolistics……………….?????? Artificial direct method of DNA transfer One of the novel physical method of exogenous DNA transfer This physical method uses accelerated micro projectiles to deliver DNA OR other molecules into intact tissues and cells Also known as GENE GUN or particle bombardment

4. Gene gun………….????? A gene gun is a device that literally fires DNA into target cells

5. Gene gun design………. The gun gene 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 , and Ted Klein of DuPont, between 1983 and 1986   expressed  the gene. The original target was onions (chosen for their large cell size) and it was used to deliver particles coated with a  marker gene . Genetic transformation was then proven when the onion tissue expressed the gene

6. Principle steps…………. Step 1: the gene gun apparatus is ready to fire Step 2 : Helium fills the chamber and pressure builds against the rupture disk. Step 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. Step 4: when the macrocarrier hits the stopping screen, the DNA-coated gold particles are propelled through the screen and into the target cells.

7. Working…… The DNA to be transformed into the cells is coated onto microscopic beads made of either tungsten or gold The coated beads are then attached to the end of the plastic bullet and loaded into the firing chamber of the gene gun An explosive force fires the bullet down the barrel of the gun towards the target cells that lie just beyond the end of the barrel

8. Conti…… When the bullet reaches the end of the barrel it is caught and stopped, but the DNA coated beads continue on towards the target cells Some of the beads pass through the cell wall into the cytoplasm of the target cells Here the bead and the DNA dissociate and the cells become transformed Once inside the target cells, the DNA is solubilized and may be expressed

9. Applications……………

10. In plants…………. This technique has been used successfully to transform soyabean, cotton, spruse, sugarcane, papaya, sunflower, rice, maize, wheat and tobacco The particle gun has also been used with pollen, early stage embroyoids, meristems and somatic embryos 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

11. Human 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 Gene gun bombardment has also been used to  transform   Caenorhabditis elegans , as an alternative to  microinjection .

12. Advantages………. Requirement of protoplast can be avoided Walled intact cells can be penetrated Manipulation of genome of subcellular organelles can be achieved 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 Relatively high efficiency Technical simplicity

13. Limitations……….. Integration is random Requirement of equipments Shallow penetration of particles Associated cell damage The tissue to incorporate the DNA must be able to regenerate Equipment itself is very expensive