Gene transfer methods, artificial, vectorless, microinjection, lipofection, liposome, electroporation

1. ARUNRAJ PT
MSc BOTANY

2. What is Gene transfer ????
“Transfer of gene from one
DNA molecule to
another DNA molecule.”

3. The transferred gene is known as Transgene and the
organism
that develop after a successful gene transfer is known
as
Transgenic.

4. The directed desirable gene transfer from
one organism to another
and
the subsequent stable integration & expression of
foreign gene into the genome
is referred as
Genetic transformation.

5. DNA TRANSFER IN NATURE

6. DNA TRANSFER IN NATURE
Vector mediated Vectorless

7. DNA TRANSFER IN NATURE
1. Conjugation
2. Bacterial transformation
3. Retroviral transduction
4. Agrobacterium mediated transfer

8. DNA TRANSFER BY ARTIFICIAL
METHODS

9. DNA TRANSFER BY ARTIFICIAL
METHODS
Vector mediated Vectorless

10. DNA TRANSFER BY ARTIFICIAL VECTORLESS
METHODS
Physical methods
1. Microinjection
2. Biolistics transformation
Chemical methods
1. DNA transfer by calcium phosphate method
2. Liposome mediated transfer
3. Transfer of DNA by use of polyethene glycol
Electrical methods
1. Electroporation

12. The microinjection is the process of transferring the desirable
DNA into the living cell ,through the use of glass
micropipette .
Glass micropipette is usually of 0.5 to 5 micrometer,
easily penetrates into the cell membrane and nuclear
envelope.
The desired gene is then injected into the sub cellular
compartment and needle is removed

13. A graphical reprasentation

16. Advantages
1. Frequency of stable integration of DNA is far better
as compare to other methods.
2. Method is effective in transforming primary cells as
well as in established cultures
3. The DNA injected in this process is subjected to less
extensive modifications

17. Limitations of microinjection
1. Costly.
2. Skilled personal required.
3. More useful for animal
cells.

19. Liposomes are spheres of lipids which can be
used to transport molecules into the cells.
These are artificial vesicles that can act as
delivery agents for exogenous materials including
transgenes.

20. Liposome- graphical representation

21. Fig:liposomes, as seen with a microscope.

22. Promote transport after fusing with the cell
membrane.
Cationic lipids are those having a positive
charge are used for the transfer of nucleic
acid.

24. Advantages
1. Simplicity.
2. Long term stability.
3. Low toxicity.
4. Protection of nucleic acid from degradation

25. Disadvantages
1. Many formulations require use of serum free, or
serum reduced medium for good efficiency
2. Some formulations are unstable to oxygen, and other
unsaturated lipids

27. Electroporation uses electrical pulse to produce
transient pores in the plasma membrane thereby
allowing DNA into the cells.
These pores are known as Electropores.

28. The cells are placed in a solution containing DNA
and subjected to electrical pulse to cause holes in
the membrane.
The foreign DNA fragments enter through holes
into the
cytoplasm and then to nucleus

31. Microscopic imaging after electroporation.
(A). Bright field image provided by confocal microscope, exhibiting
some abnormal protrusion on the cell surface.
(B). Same visual fields were placed under specific exciting light for
Gfp emission observation. Scale bars: all are 30μm.

32. 1. Method is fast.
2. Less costly.
3. Applied for a number of cell types.
4. Simultaneously a large number of cell
can be treated.
5. High percentage of stable transformants
can be produced
Advantages of Electroporation

33. 1. Chance for cell damage
Disadvantages of Electroporation

34. CONCLUSION
 Gene transfer is a way to integrate a
foreign gene to a genome.
 Its a recent approach in biological science.
 Transfer is categorized into:
1. Gene transfer by Natural way and
2. Artificial methods.

35.  Natural gene transfer categorized into
two: vectorless and vector mediated
 Similarly, artificial methods to vectorless
and vector mediated.
 Artificial transfer has been sub
categorized into: Physical, chemical and
Electrical.

36. REFERENCES
I. Chawla, H.S. 2000. Introduction to plant
biotechnology. Oxford & IBH
publishing Co. Pvt. New Delhi
II. Gupta, P.K. 1999. Elements of
Biotechnology. Rastogi Publications.
Meerut
III. Griffiths et al, 1999. Modern Genetic
Analysis. W.H. Freeman & Co. New
York