GENE TRANSFER TECHNIQUES: TRANSFECTION

SAHANA V

PG DIPLOMA

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.

Transformation and Transfection??

Same!!

Difference???

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

Transfection

Non- Viral

Mechanical

1. Microinjection2. Particle Bombardment

3. Single walled Carbon nano-tubes

Physical

1. Electroporation2. Liposome mediated

3.Polymers4. Dendrimers

Viral

1. Viruses2. Virus Like

Particles

VIRUSES IN USEViral 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

FOOT NOTES

100% efficiency Toxicity Strong immune response Untargeted integration of genes Complexity of generating recombinant

viruses Limited packaging capabilities In vivo DNA delivery

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

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.

SWNT

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)

PAMAM DENDRIMERS

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.