3. Verified by Vaheri and Pagano in 1965.
Principle:
DNA is mixed with DEAE-dextran.
DNA/polymer complex comes into contact with
negatively charged membrane due to excess of positive
charge contributed by polymer.
Uptake presumably by endocytosis.
DEAE (Diethylaminoethyl)-dextran
5. Advantages:
It is very simple method
Low cost
Disadvantage:
Transfection efficiency is low
DEAE (Diethylaminoethyl)-dextran
6. Verified by Graham and Van in 1973.
Principle:
DNA is placed in phosphate buffer.
Calcium chloride solution addition in DNA and phosphate
buffer.
Formation of DNA-calcium phosphate coprecipitates which
adhere to surface of cells.
Uptake presumably by phagocytosis.
Calcium-Phosphate coprecipitation
8. Advantages:
Components are easily available and reasonable in price.
Generate stably transfected cell lines, allowing for long-term
gene expression studies.
Disadvantages:
Its sensitive to slight changes in pH, temperature and buffer
salt concentrations.
Expression is dependent on cell division after transfection.
Calcium-Phosphate coprecipitation
9. Liposome mediated gene transfer
Lipofection is the most commonly used chemical gene
transfer method.
Cationic transfection lipids consist of:
A positively charged head group such as an amine
A flexible linker group such as an ester or ether
And two or more hydrophobic tail groups
Lipofection
11. Principle:
A cationic lipid is mixed with a neutral lipid/helper lipid,
liposome vesicles are formed carrying a net positive charge.
Nucleic acids adsorb to these vesicles.
Ionic absorption to the cellular membrane.
Uptake presumably by endocytosis.
Neutral lipids allow entrapped DNA to escape into cell by
fusion of the lipsome with the membrane.
Lipofection
13. Advantages:
Transfect a wide range of cell types
Less costly
Successful delivery of DNA of all sizes
Disadvantage:
Low efficiencies in most primary cells
Lipofection
14. Used for protoplast only.
PEG stimulates endocytosis therefore DNA uptake
occurs.
Protoplasts are kept in PEG solution.
After DNA transfer PEG and other chemicals are
removed.
Polyethylene glycol mediated transfection
15. 1. Identify a suitable explant
2. Co-cultivate with the Agrobacterium
3. Kill Agrobacterium with antibiotic
4. Select for transformed plant cells
5. Regeneration of whole plant
Agrobacterium mediated Transformation
17. It is used for manipulation of single cells, such as oocytes
by injection of DNA, mRNA, and proteins.
It can also be used for the transfer of DNA into embryonic
stem cells to generate transgenic organisms.
Principle:
Using a micromanipulator and a very fine tipped
micropipette having 0.5 to 5 micrometer size is inserted
into the cytoplasm or directly into the nucleus.
Microinjection
19. Advantage:
High efficiency (nearly 100% efficient)
Disadvantages :
Requires certain operator skills
Very time consuming and expensive
Microinjection
20. Used to deliver nucleic acid to cultured cells as well as to
cells in vivo.
Principle:
Transfer of DNA coated on the surface of micro particles
such as gold or tungsten.
Particles are accelerated by a particular driving force, e.g. by
establishing a high voltage discharge between two
electrodes or gas pressure.
Biolistic Particle Delivery
22. Advantages:
Technique is fast and simple
Transfection of dividing and non-dividing cells
No limit to the size or number of genes that can be delivered.
Disadvantages:
Mortality is very high and therefore need high cell numbers.
Biolistic Particle Delivery
23. Electroporation is a frequently used physical gene transfer
method.
Principle:
Cells and DNA are suspended in an electroporation buffer.
High-voltage pulses of electricity are applied to the cells.
Electrical pulse creates a potential difference across the
membrane.
Induces temporary pores in the cell membrane for DNA entry.
Electroporation