Direct gene transfer methods.pptx including types

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1. Direct Gene Transfer Methods II BSc Biotechnology

2. I NTRODUCTION It is defined simply as a technique to efficiently and stably introduce foreign genes into the genome of target cells. These methods are also classified in two groups : Direct method and Agrobacterium mediated gene transfer. Direct Gene transfer relies on the delivery of large amounts of DNA while the plant cell is transiently permeabilized by Particle bombardment, Electroporation, ultrasound, etc. It is mostly used for gene transfer in cereal crops ( monocotyledonous and dicotyledonous crops). Agrobacterium mediated gene transfer are based on utilizing Agrobacterium, a pathogen of dicotyledonous (broad leafed) plants that transfers genes into the plant genome. 3

3. 4 6/8/2017 G ENE C LONING The general approach for genetic engineering in plants may be outlined as follows: Introduction of the gene of interest into the cells of concerned plant species. Integration of this gene into the nuclear/organellar genome of the plant cells. Expression of the trassferred gene in the new genetic background. Regeneration of whole plants from the genetically modified cells and Transmission of the transferred gene to the sexual progney of these plants. It may be noted that gene transfers in plants are primarily based on tissue culture and that the itetgration and expression of the produced genes must be stable to be transmitted through the sexual process.

4. Plant Transformation Methods Ph y sical Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Silica/carbon fibers Lazer mediated Chemical PEG Calcium phosphate Artificial lipids Proteins Dendrimers Biological A. tumefaciens A. rhizogenes Virus-mediated Direct Method 6/8/2017 G ENE C LONING 5

5. 6/8/2017 G ENE C LONING 6 Introduction of DNA into plant cell by exposing them for very brief periods into high voltage electric pulses, which induce transient pores in the plasma lemma, is called ELECTROPORATION. Two systems: Low voltage and long pulse method: 300- 400 V/cm for 10-50 milliseconds. h i gh volt a n d s hort p u l se m e t ho d : 100 - 1500 V/ c m for 10 microseconds (more stable). Transformation frequency are increased several folds by A heat shock to protoplast just prior to electroporation (5 mins at 45°C). The pr e sence o f l ow c once n tr a t i on o f PEG ( 8 % ) dur i nd electroporation. Electroporation

6. S TEPS Advantage : Some plants are PEG sensitive so electroporation may be of choice, m ethod is fast, less costly and high percentage of stable transformants can be produced. Application : it has been used to produce stably transformed cell lines and plants of several species like tobacco, maize, rice, wheat, etc. In tobacco frequency is as high as 2-8% in presence of 7% PEG.

7. M I CRO INJECTION Microinjection is the method tried for artificial DNA transfer to cereals plants that show inability to regenerate and develop into whole plants from cultured cells. Around 0.3 ml of DNA solution is injected at a point above tiller node until several drops of solution came out from top of young inflorescence. Timing of injection is important and should be fourteen days before meiosis . Method has been successfully used with cells and protoplast of tobacco, alfalfa etc. Fine tip is used (0.5 - 1.0 micrometer diameter) glass needle or micropipette. 6/8/2017 G ENE C LONING 8

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9. A DVANTAGES LIMITATIONS OF BIOLISTICS 6/8/2017 G ENE C LONING Advantages 1. Requirement of protoplast can in t act cells ca n be be avoided. 2. Walled penetrated. 3. M a nipulati o n of geno m e of be sub c ellular o r ganelles can achieved. Limitations Shallow penetration of particles Associated cell damage The inability to deliver the DNA systemically The tissue to incorporate the DNA must be able to regenerate And the equipment itself is very expensive. 16

10. LIPOSOME MEDIATED GENE TRANSFER Liposomes are spheres of lipids which can be used to transport molecules into the cells. It is called lipofection. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. They are considered as sphere of lipid bilayers surrounding the molecule to be transported and promote transport after fusing with the cell membrane. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid. These liposomes are able to interact with the negatively charged cell membrane more readily than uncharged liposomes, with the fusion between cationic liposome and the cell surface resulting in the delivery of the DNA directly across the plasma membrane. Cationic liposomes can be produced from a number of cationic lipids, e.g. DOTAP and DOTMA. These are commercially available lipids that are sold as an in vitro-transfecting agent, as lipofectin. Liposomes for use as gene transfer vehicles are prepared by adding an appropriate mix of bilayer constituents to an aqueous solution of DNA molecules. The liposomes are then ready to be added to target cells. Germline transgenesis is possible with liposome mediated gene transfer and ES cells have been 6/8/2017 successfully tr G a E n NE s C fe LO c N t IN e G d by liposomes also. 18

11. A DVANTAGES 1. Simplicity. 2. Long term stability. 3. Low toxicity. 4. Protection of nucleic acid from degradation 19 6/8/2017 G ENE C LONING

12. POLYETHYLENE GLYCOL MEDIATED TRANSFECTION 6/8/2017 G ENE C LONING 20 This method is utilized for protoplast only. Polyethylene glycol stimulates endocytosis and therefore DNA uptake occurs. Protoplasts are kept in the solution containing PEG. Calcium chloride is added and sucrose and glucose acts as osmotic buffering agent. After exposure of the protoplast to exogenous DNA in presence of PEG and other chemicals, PEG is allowed to get removed. Intact surviving protoplasts are then cultured to form cells with walls and colonies in turn. After several passages in selectable medium frequency of transformation is calculated. PEG based vehicles were less toxic and more resistant to nonspecific protein adsorption making them an attractive alternative for non-viral gene delivery.

13. 21 Plant protoplast are suspected in the transformation medium rich in Mg 2+ ions. Linearized plasmid DNA containing the gene construct is added to the protoplast suspension Add 20% PEG and ph of medium 8. Give heat shock for 5 min at 45 °C. Transfer in ice. 6/8/2017 G ENE C LONING

14. DNA TRANSFER BY DAE-DEXTRAN METHOD 6/8/2017 G ENE C LONING DNA can be transferred with the help of DAE Dextran also. DAE-Dextran may be used in the transfection medium in which DNA is present. This is polycationic, high molecular weight substance and convenient for transient assays in cos cells. It does not appear to be efficient for the production of stable transfectants. If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell can express the transferred gene. It is known that serum inhibits this transfection so cells are washed nicely to make it serum free. Stable expression is very difficult to obtain by this method. Treatment with chloroquinine increases transient expression of DNA. The advantage of this method is that, it is cheap, simple and can be used for transient cells which cannot survive even short exposure of calcium phosphate. 22

15. CALCIUM PHOSPHATE MEDIATED DNA TRANSFER 6/8/2017 G ENE C LONING The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate under condition which allow the precipitate of calcium phosphate to be formed. Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis. Transfection efficiencies using calcium phosphate can be quite low, in the range of 1-2 %. It can be increased if very high purity DNA is used and the precipitate allowed to form slowly. Limitations of calcium phosphate mediated DNA transfer Frequency is very low. Integrated genes undergo substantial modification. Many cells do not like having the solid precipitate adhering to them and the surface of their culture vessel. Due to above limitations transfection applied to somatic gene therapy is limited. 23