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  1. 1. Presented by,Presented by,Presented by,Presented by, Priyanka Sarkar Roll No:Roll No:Roll No:Roll No:----16161616 M.Sc. 3M.Sc. 3M.Sc. 3M.Sc. 3rdrdrdrd Sem,2010Sem,2010Sem,2010Sem,2010
  2. 2. J. Bertram,2005 • Used magnetic particles to improve delivery of Biomolecules, including DNA, into cells. • protein production for clinical or research applications • changing the protein expression profile of a cell to assay for the effects of a gene viz. Over expression of gene RNAi-reduction in expression of gene • addition of genetic markers to a cell line
  3. 3. The process of transferring foreign DNA fragments into a recipient (host) cell for growth and replication. Bacteria must be “competent" for the uptake of foreign DNA fragments. ◦ produce specialized proteins◦ produce specialized proteins ◦ competence may be triggered heat shock or CaCl2 treatment DNA segments must be small ◦ ~ 500 kb
  4. 4. plasmidplasmid Gene of interest
  6. 6. GOIGOIGOIGOI incorporatedincorporatedincorporatedincorporated
  7. 7. 1. The plasmid vector must be cut with a restriction endonuclease (aka: restriction enzyme). 2. DNA ligase joins the DNA fragment & vector DNA 3. Host cell is made competent so that plasmid can enter.enter. 4. Transformed cells are grown on selection media . 5. The transformation system includes 3 components viz. mode of delivery, selectable marker and propagation of clones.
  8. 8. Cold Shock with high concentrations of calcium chloride and magnesium chloride make the cells competent. For preparation of comp cells..For preparation of comp cells..For preparation of comp cells..For preparation of comp cells.. Grow cells in SOB medium Transformation Methods: 1. Chemical treatmentChemical treatmentChemical treatmentChemical treatment Grow cells in SOB medium Cells treated with high conc.of chemicals Immediate cold shock at -1960C. store at -800C and use for transformation
  9. 9. The bacterial cells are taken into the mouse and given shock of 2.5 mV. Electric Shock Opens Pores in Cell WallWall
  10. 10. Shoots projectiles of gold or tungsten coated with DNA or RNA into cells Generally used with Eukaryotes
  11. 11. Incase of plants it is either Non Integrative:- in it the transgene is maintained stably in an extrachromosomal state but not transfer throughout the generation. e.g.:- virus vectore.g.:- virus vector Integrative:- in it the foreign gene integrated into the host gene and so can pass onto the next generation. e.g.:- A variety of process used to introduced transgene into plant cells, Can be grouped into 2 categories:- 1.Indirect gene transfer And 2.Direct gene transfer
  12. 12. Gene transfer through Agrobacterium. These occur in 2 ways:- a. co-culture with tissue explants && b. In plant transformation
  13. 13. By placing viral genome into the T-DNA of a Ti-plasmid Agrobacterium used for co-culture with the plant cell Used with such viruses which have to be transmitted by an insect vector Used for successful infection.Used for successful infection. e.g. geminiviruses,viz. Maize RS streak virus & Wheat dwarf virus
  14. 14. Introduction of foreign DNA without involvement of a biological agent. Various methods are as follows:- a> chemical methodchemical methodchemical methodchemical method:- by using certain chemicals. b> electroporationelectroporationelectroporationelectroporation:- Introduction of DNA into cell by exposing them for some period to electrical pulses.period to electrical pulses. .it induce transient pores in the plasma membrane. Basically 2 systems are there- 1.Low voltage-long pulse method- produces transient transformation 2.High voltage-short pulse method- produces stable transformation c> Particle Gun MethodParticle Gun MethodParticle Gun MethodParticle Gun Method -1-2micro m tungsten & gold particle DNA to be
  15. 15. DNA Vacuum DNA
  16. 16. LASER−INDUCED DNA DELIVERY It is used to deliver DNA into plant cell. Lasers puncture transient holes in the cell membrane. Used for high transfection freequency (10 3).Used for high transfection freequency (10 3). There is little information on transient expression or stable integration.
  17. 17. RefferencesRefferencesRefferencesRefferences Gene cloning –T.A.Brown Genes and Genome- Richard J. Reece.
  18. 18. Collage on Photostory Dolly