Gene transfer methods

Gene transfer methods, rDNA Technology

1. Gene transfer techniques By, Abhinava J V BIRAC Innovation Fellow University of Agricultural Sciences, Dharwad

2. Direct DNA transfer

3. Physical gene transfer methods H igh voltage electrical impulses are used to create the pores in the plasma membrane. T hrough which DNA enters and gets integrated into the host cell genome. 1 . Electroporation :

4. There are other names for this technique- particle gun, gene gun, bio blaster. DNA is coated with the tungsten or gold particles called as Micro carriers (micro projectiles) These are carried by macro carriers (macro projectiles). These macro-carriers are inserted into the apparatus and pushed downward by rupturing the disc . The stopping plate does not permit the movement of macro carrier while the micro carriers (with DNA) are propelled at a high speed into the plant material . 2. Particle Bombardment:

5. The technique of microinjection involves the transfer of the gene through a micropipette (0.5-10.0 pm tip) into the cytoplasm/nucleus of a plant cell or protoplast. The recipient cells are kept immobilized in agarose embedding, and held by a suction holding pipette. 3. Microinjection :

6. Chemical Gene Transfer Methods: Liposomes are artificially created lipid vesicles containing a phospholipid membrane. They are successfully used in mammalian cells for the delivery of proteins, drugs etc. Liposomes carrying genes can be employed to fuse protoplasts and transfer the genes . The efficiency of transformation increases when the process is carried out in conjunction with polyethylene glycol ( PEG). 1. Liposome-Mediated Transformation :

7. The desirable DNA can be complexed with a high molecular weight polymer diethyl amino ethyl (DEAE) dextran and transferred. 2. DEAE Dextran-Mediated transfer: 3 . Calcium Phosphate Co- Precipitation-Mediated Transfer : The DNA is allowed to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate. When the actively dividing cells in culture are exposed to this precipitate for several hours, the cells get transformed.

8. Indirect DNA transfer

9. Agrobacterium-Mediated Gene Transfer Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium. It is rod shaped and motile, and belongs to the bacterial family of Rhizobiaceae . A. tumefaciens contain a 200-kb tumor inducing (Ti) plasmid Bacteria was capable to transfer a portion of the plasmid DNA into the plant host (T-DNA). Infects plants through breaks or wounds. Expression of genes in wild-type T-DNA results in dramatic physiological changes to the plant cell. T-DNA Synthesis the of plant growth hormones ( auxins and cytokinins ) which cause to neoplastic growth (tumor formation)

10. Agrobacterium tumefaciens chromosomal genes: chvA , chvB , pscA required for initial binding of the bacterium to the plant cell and code for polysaccharide on bacterial cell surface . Virulence region ( vir ) carried on pTi , but not in the transferred region (T-DNA). Genes code for proteins that prepare the T-DNA and the bacterium for transfer. At least nine vir -gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1, D2 and D4, and vir E1, and E2.

11. Virus-Mediated Gene Transfer: Mono-partite viruses: e.g. tobacco mosaic virus (TMV). Multipartite viruses : , e.g. brome mosaic virus (BMV) 1. DNA Based: Cauliflower mosaic virus ( CaMV ) : Gemini Viruses as Vectors: 1. R NA Based:

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