Various Techniques of DNA introduction in target organs

1. DNA RECOMBINANT TECHNOLOGY
Topic: Various techniques of DNA introduction in
target organs
Maryam Saddiqa

2. Chemical Methods
 DEAE (Diethylaminoethyl)-dextran
 Calcium-Phosphate coprecipitation
 Lipofection
 Polyethylene glycol (PEG)
Physical Methods
 Biolistic Particle Delivery
 Microinjection
 Electroporation
Biological methods
 Agrobacterium mediated transformation
Transformation Methods

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

4. 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

7. 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

10. Cationic lipid

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

12. 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

16. 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

18. 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

21. 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

24. Electroporation

25. Advantages:
 Method is fast
 Less costly
 Large number of cells treated simultaneously
Disadvantage:
 High mortality rates
Electroporation