Vector less gene transfer

Vector less gene transfer

1. SAAKRE MANJESH 2014-11-105 Vector less gene transfer/Direct gene transfer methods

2. Gene transfer Indirect gene transfer/ Agrobacterium mediated Direct gene transfer/ Vector less gene transfer 1. Chemical methods- PEG CaPO 4 precipitation DEAE-Dextran mediated Liposome mediated 2. Physical methods - Electroporation Microinjection Gene gun 3. Imbibition

3. Direct gene transfer Foreign gene of interest is delivered into the host plant cell without the help of a vector . Introduction of DNA into plant cells with out the involvement of a biological agent, e.g., Agrobacterium leading to stable transformation. Stable method of gene transfer Different chemical and physical treatments are employed to facilitate the entry of DNA into plant cells

4. Chemical mediated gene transfer

5. Protoplasts are treated with a solution containing various ions, PEG and DNA. Changes in the plasma membrane allow the DNA to penetrate and move into the cytoplasm. Whether PEG is directly involved in the delivery of DNA and the mechanism of this process are still unclear The integration of the target DNA into the plastid chromosome is site-directed due to the design of vectors with sequences homologous to a specific target area in the plastid genome. PEG Mediated gene transfer

6. Plant protoplasts are suspended in a transformation medium rich in Mg2+ Linearized plasmid containing gene/DNA is added PEG is added, adjust the PH about 8 Give 5 min heat shock at 45°C followed by transfer to ice procedure

7. Advantages Yield more than 3% transformation More reliable and efficient Frequency of transformation is high, if we use 2-7% PEG Disadvantages Plant cells are most sensitive to PEG

8. Lipofection Introduction of DNA into cells via liposomes Transformation frequencies of 4 Χ 10 -5 Plasmid DNA of 9kb and larger DNA can be transferred Higher transformation frequencies with PEG and electroporation make them more active

9. Mechanism

10. Advantages: High efficiency Its ease of use Reproducibility Low toxicity Dis Advantages Not applicable to all cell types

11. Calcium phosphate precipitation Mixing of DNA in phosphate buffer solution , adding this in controlled manner to a calcium chloride solution and allowing the mixture to incubate at room temperature This step generates a precipitate that is dispersed onto the cultured cells The precipitate is taken up by the cells via endocytosis or phagocytosis

12. Calcium phosphate precipitation Optimal final conc. Of DNA in precipitate mixture is 20 μ g/l

13. DEAE-Dextran-mediated Transfection DEAE-Dextran ( diethylaminoethyl -dextran) is water soluble and polycationic Added to the transfection solution containing the DNA Brings about DNA uptake b the cells through endocytosis Interaction with – vely charged DNA and with the components of cell surface plays an important role. Disadvantage: Not efficient in producing stable transformation

14. Physical methods of gene transfer

15. Microinjection The DNA solution is directly injected directly inside the nucleus of a cell It uses capillary glass micropipettes with the help of micromanipulators of microinjection assembly Success has been claimed in the transfer of a gene for resistance to the antibiotic kanamycin.

16. Conti…. The protoplasts are immobilized on glass slides/ petriplates coated with polylysine or by holding them under suction by a micropipette Agar used as solidifying agent High costly equipments Successful in Brassica napus (microspore cells), Barley

17. Microinjection assembly Stereoscopic dissecting microscope Microinjector

18. Electroporation Mix the DNA with protoplasts containing suitable ionic solution Expose high voltage electrical pulses for very brief periods of time (300-400 V/cm for 10-50 ms ) It induce transient pores in the plasma lemma called Electroporation

19. Advantages Stable transformed cells Easy to perform Disadvantages Applicable for only sensitive plants Low copy number

20. Fibre mediated DNA delivery Suspension culture cells were mixed with DNA Silicon carbide fibres of 0.6 μ m dia and 10-80 μ m length The mixture should be vortexed DNA-coated fibers penetrate the cell wall in the presence of small holes created in collisions between the plant cells and fibers.

21. Advantages Easy and quick procedure Low  expenses and  Usefulness for various plant materials Dis advantages low transformation efficiency Cause damage to cells 

22. Laser Induced DNA Delivery The plasma membrane of a cell is then exposed to a laser beam for a small amount of time The generation of a photopore allows exogenous  DNA Laser puncture transient holes in the cell membrane through which DNA may enter into the cell cytoplasm High transformation frequency Low stable integration

23. Direct gene transformation through imbibition U ptake of exogenous DNA of dehydrated plant tissues presence of 20% DMSO , suggesting that membrane permeability was an important factor in the process . D essicated somatic embryos of alfalfa, showed transient GUS expression at frequencies up to 70 %. The stable transformation of rice by embryo imbibition was also reported.

24. Advantages Most simple of methods , as they require no specialist equipment preparation of target plant tissues generally simple Dis advantages T hey can be applied only to very specific organs or tissues (i.e. newly pollinated flowers or hydrating embryos ) It is still not clear that they lead to stable , and heritable transformation.

25. Dendrimer mediated gene transfer + vely charged amino groups on the surface of the dendrimer molecule interact with the – vley charged DNA to form DNA- dendrimer complex DNA- dendrimer complex ha overall positive charge and can bind negatively charged surface molecules on membrane of eukaryotic cells Complexes bound to the cell surface are taken into the cell by nonspecific endocytosis The complexes are transferred to the endosomes

26. References Text book of Biotechnology expanding horizon, By B.D Singh Text book of Life Scineces fundamentals and practice part II by Pranav kumar and Usha mina http://www.scribd.com/doc/54262070/Direct-Gene-Transfer-Methods#scribd http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10-0826_transfection_tutorial_interactive.pdf

27. Thank you