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GENE TRANSFER TECHNIQUES

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GENE TRANSFER TECHNIQUES

  1. 1. GENE TRANSFER TECHNIQUES: TRANSFECTION SAHANA V PG DIPLOMA
  2. 2. TERMINOLOGY  Transfection : Introduction of foreign DNA into eukaryotic cells usually animal cells.  Transfectants: Cells that have incorporated foreign DNA.  Stable : Integrated foreign DNA.  Transient: Does not integrate foreign DNA, but genes are expressed briefly.
  3. 3. DIFFERENCE Cloning (uptake of genetic material) Cancer Transformation Transfection Transformation In unicellular organisms like prokaryotes (bacteria) or unicellular eukaryotes (amoeba) In Metazoan Eukaryotic Cells Advancement of a metazoan eukaryotic cell from being non- cancerous to cancerous
  4. 4. Transfection Non- Viral Mechanical 1. Microinjection 2. Particle Bombardment 3. Single walled Carbon nano- tubes Physical 1. Electroporation 2. Liposome mediated 3.Polymers 4. Dendrimers Viral 1. Viruses 2. Virus Like Particles
  5. 5. VIRUSES IN USE Viral Vector DNA Insert Size Expression Pitfalls Retro viral 8 kb Stable Random insertion site Lenti viral 9 kb Stable Random insertion site Adeno Virus 8 kb Transient Highly immunogenic Adeno associated Virus 5 kb Stable, site specific location Requires helper virus and difficult to remove Herpes Simplex Virus 30-40 kb Transient No gene expression during latent infection Vaccina Virus 25 kb Transient Potential cytopathic effects
  6. 6. FOOT NOTES  100% efficiency   Toxicity   Strong immune response   Untargeted integration of genes   Complexity of generating recombinant viruses   Limited packaging capabilities   In vivo DNA delivery 
  7. 7. VIRUS LIKE PARTICLES(VLP)  Alternative approach to classical methods  Viral capsid- without viral genetic information.  Eg: Pappilloma viruses: L1 and L2 proteins  Predominantly use – vaccination  Gene delivery – human polymo JC virus, murine polymovirus, pappilomaviruses and AAV- based VLPs. Isolation and purification of viral capsid proteins Empty viral particles reconstituted and stored at -80 0C Packaging with DNA or siRNA inside empty viral particles
  8. 8. SINGLE WALLED CARBON NANOTUBES – MECHANICAL METHOD  Unidimensional layer of carbon-hexagons form a tube.  Functionalized- amino or carboxyl group  Covalent or non-covalent bond with biomolecules.  Diameter: 1-5nm; Length: 50-200nm  Success: In vivo siRNA delivery.
  9. 9. SWNT
  10. 10. PAMAM DENDRIMERS- PHYSICAL METHOD  Polyamidoamine (PAMAM)- non-linear polycationic cascade- binds plasmid DNA  Activated PAMAM + plasmid DNA = condensation of nucleic acid  Compact transfection complex – adhere to cell surface and taken up by endocytosis  Generation 6/7 with 6 & 10nm – gene transfer  Commercially available – SuperFect transfection reagent and PolyFect transfection reagent (QIAGEN, Germany)
  11. 11. PAMAM DENDRIMERS
  12. 12. SUMMARY  Chemical methods : system needs to be adapted to the cargo to be delivered.  No separate genetic protocols for siRNA and plasmid DNA delivery.  Physical methods : cytotoxicity, cellular uptake insufficient mostly.  What is needed?  Specific tailor-made DNA and siRNA delivery systems  Nucleic acid-based therapeutics : individualized medicines for specific disease variation.

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