This document discusses various gene transfer techniques called transfection. Transfection is the introduction of foreign DNA into eukaryotic cells, producing transfectants that have incorporated the DNA. Stable transfectants integrate the DNA while transient transfectants express genes briefly without integration. Methods include mechanical techniques like microinjection and bombardment, physical techniques like electroporation and liposomes, and viral vectors. Viruses commonly used include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses. Non-viral methods discussed are virus-like particles, single-walled carbon nanotubes, and polyamidoamine dendrimers. Overall, the document compares techniques and notes what is needed

1. GENE TRANSFER
TECHNIQUES: TRANSFECTION
SAHANA V
PG DIPLOMA

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.

4. 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

5. 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

6. 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

7. FOOT NOTES
 100% efficiency 
 Toxicity 
 Strong immune response 
 Untargeted integration of genes 
 Complexity of generating recombinant viruses 
 Limited packaging capabilities 
 In vivo DNA delivery 

8. 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

9. 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.

10. SWNT

11. 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)

12. PAMAM DENDRIMERS

13. 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.