Gene transfer methods

GENE TRANSFER METHODS

1. GENE TRANSFER METHODS SUBMITTED BY ATHIRA S II MSc DEPT OF BIOCHEMISTRY

2. INTRODUCTION  Gene transfer or uptake of DNA refers to the process that moves a specific piece of DNA into cell. The directed desirable gene transfer from 1 organism to another & the subsequent stable integration & expression of foreign gene into the genome is referred as genetic transformation. The transferred gene is known as transgene & the organism that develop after a successful gene transfer is known as transgenic .

3. METHODS OF GENE TRANSFER

4. BIOLOGICAL METHODS

5. CHARACTERISTICS OF CaMV VIRUS : dS DNA virus. Packaged as nucleosome . -Mechanical and aphid mediated transmission - Virion DNA alone or cloned CaMV DNA is infectious when simply rubbed on leaves -Up to 10 6 copies per cell. 3-4 weeks for systemic infection through plant.

6. Results of gene transfer plants- Use of vectors based on the gemini tomato golden mosaic virus (TGMV) to introduce the neomycin phosphotransferase ( neo gene) into cereals, cassava plants . {*Source: Nature 334 , 179 - 182 (14 July 1988)}

7. PHYSICAL METHODS

8. 3.ELECTROPORATION An electric field mediated-high voltage electric pulse is implicated for the fusion of cells-plasma membrane in this method. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution. This is an simple and rapid technique for introducing of genes into the cells of various organisms (microorganisms, plants and animals). Electroporation is an effective way to transform E.coli cells containing plasmids with insert DNAs longer than 100 kb. The transformation efficiency is around 109 transformants per microgram of DNA for small plasmids (about 3kb) and about 106 for large plasmids (about 130 kb).

9. Method of Electroporation : under the influence of electric shocks, holes will create in the plasma membrane, through which foreign DNA fragment enter into the host cytoplasm and after that into the nucleus

10. 4. Liposome-Mediated Gene Transfer Liposomes are small circular lipid molecules carry nucleic acid. Such molecules easily stick to the membrane of the host cell and transfer DNA fragments into the nucleus.

11. 6.DIRECT TRANSFER OF DNA Direct DNA transfer methods are those methods which do not utilize bacteria as intermediaries for incorporation of DNA into host genome.,through these methods foreign DNA fragment inject directly in the nucleus of the host plant cell. Microinjection, particle bombardment ( biolistics or Microprojectile ) methods are the some important techniques through which DNA directly injected into the genome of transformed cell

12. MICROINJECTION Direct mechanical introduction of DNA under microscopical control in specific target. Microinjection is able to penetrate intact cell wall. Host range independent and does not require a protoplast regeneration system. Cells/protoplast-glass micropipette of 0.5-10.0 µm diameter tips are used for transfer of macromolecule into the cytoplasm/nucleus of recipient cell/protoplast. Recipient cells can be immobilized by using methods such as agarose embedding, agar embedding, poly-lysine treated glass surface & suction holding pipette.

13. Once injection achieved, the injected cell must be cultured properly to ensure its continued growth and development. Disadvantage- production of chimeric plant with only a part of plant is transformed. Process slow, expensive, requires highly skilled and experienced personnel. Injection of DNA solution (5-10 µl) by micropipettes into the developing floral side shoot (tillers) of plant . Within the floral tillers are achesporial cells that give rise to pollen in the developing sac by two meiotic cell division. Such cells might also be able to take up large molecules such as DNA.

14. PARTICLE BOMBARDMENT /BIOLISTIC / PARTICLE GUN METHOD Biolistic is process of bombarding cells with microscopic projectile coated with DNA . Shot at high velocity from particle gun into cells/tissue. Promising for plant which can not be infected by Agrobacterium . DNA delivery to plant cell made possible when heavy microparticle or microcarrier (tungsten/gold) coated with the DNA of interest are accelerated to very high initial velocity are made to bombard the living plant cell. Microparticle penetrate the cell wall & get integrated into the plant genome. High frequency of integration of multicopy insertion; no regeneration protocol neces

15. DNA coating is sophisticated technology & requires precise preparation of DNA coated gold/tungsten particle. Gold- uniform size & shape, less toxic to cells. Coating of micropellet with DNA by precipitation is important step. 1.25 to 18 mg microparticles are mixed with 0.5 to 70 µg of plasmid DNA in CaCl2 (0.25 – 2.5 M) and spermidine (0.1 M) solution. Mixture continuously vortexed to ensure uniform coating After DNA precipitation, micropellets palced on macrocarrier membranes & allowed to dry and immediately used.

16. Microprojectile bombardment or biolistic -mediated DNA transfection equipment (a) lab version (b) portable version When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk * with the DNA-coated gold particles on its lower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.

17. Microprojectile bombardment ( biolistics ) apparatus

18. CHEMICAL METHODS Involves plasma membrane destabilizing &/or precipitating agents. Protoplast are mainly incubated with DNA in buffer containing PEG, Poly L- ornithine , polyvinyl alcohol or divalent ions.

19. PEG MEDIATED GENE TRANSFER First conclusive demonstration of uptake & integration of isolated Ti plasmid DNA into plant protoplast was reported in Petunia & tobacco in the presence of PEG/Poly L- ornithine . Protoplast are isolated- particular concentration of protoplast suspension taken in tube- followed by addition of plasmid DNA. To this 40% PEG 4000 (w/v) dissolved in mannitol & calcium nitrate solution added slowly because of high viscosity & mixture incubated for few minutes. Advantage: form of the DNA applied to the protoplasts is controlled entirely by the experimenter and not by an intermediate biological vector. Disadvantage: system requires protoplast & a functional system for regeneration of theses protoplast to calluses & whole plant.

20. CONCLUSION Physical methods for transferring of genes are more effective for single/ multiple targeted cells. This method carries modest risk of transfection reagents. Further through these methods scientist observed results very quickly and in short span of time. However they also have some drawbacks. For example, it is very difficult for the genes to be transported into the nucleus because of little access in passing through the membrane or enzymatic degradation of the naked DNA or RNA, which resulted low transfection efficiency and low clinical applications. On the other hand, they present damage to cells; difficulty has been seen in large-scale manipulation, labor -intensive protocols and very high cost of the instruments, which only available in well develop labs.

21. REFERENCES Biotechnology – by U.Satyanarayana A textbook of biotechnology – R.C.Dubey