This document discusses various techniques for gene transfer, including natural methods like conjugation, transformation, and transduction, as well artificial methods like microinjection, biolistics, calcium phosphate and liposome mediated transfer, and electroporation. It provides details on how each method works, such as how conjugation involves transfer of DNA between bacteria via sex pili, and how electroporation uses electrical pulses to create pores in cell membranes to allow DNA entry. The document also summarizes screening and applications of transgenic techniques.

1. GENE TRANSFER
TECHNIQUES
MANDAWI TRIPATHI

2. INTRODUCTION
• Gene transfer is to transfer a gene from one DNA molecule to
another DNA molecule.
• 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.
• Transient transformation occur when DNA is not integreted
into host genome

3. • Stable transformation occur when DNA is integrated into host
genome and is inherited in subsequent generations.
• The transferred gene is known as transgene and the organism
that develop after a successful gene transfer is known as
transgenic.

4. METHODS OF GENE TRANSFER
DNA transfer by natural methods
• 1. Conjugation
• 2. Bacterial transformation
• 3. Retroviral transduction
• 4. Agrobacterium mediated transfer

5. DNA TRANSFER BY ARTIFICIAL METHODS
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Physical methods
1. Microinjection
2. Biolistics transformation
Chemical methods
1. DNA transfer by calcium phosphate method
2. Liposome mediated transfer
Electrical methods
1. Electroporation

6. CONJUGATION
• Requires the presence of a special plasmid called the F
plasmid.
• Bacteria that have a F plasmid are referred to as as F+ or
male. Those that do not have an F plasmid are F- of female.
• The F plasmid consists of 25 genes that mostly code for
production of sex pilli.
• A conjugation event occurs when the male cell extends his
sex pilli and one attaches to the female.

7. • This attached pilus is a temporary cytoplasmic bridge
through which a replicating F plasmid is transferred from
the male to the female.
• When transfer is complete, the result is two male cells.
• When the F+ plasmid is integrated within the bacterial
chromosome, the cell is called an Hfr cell (high frequency
of recombination cell).

9. TRANSFORMATION
• transformation is the direct uptake of exogenous DNA from its
surroundings and taken up through the cell membrane .
• Transformation occurs naturally in some species of bacteria,
but it can also be effected by artificial treatment in other
species.
• Cells that have undergone this treatment are said to be
competent.
• Any DNA that is not integrated into he chromosome will be
degraded.

11. TRANSDUCTION
• Gene transfer from a donor to a recipient by way of a
bacteriophag..
• If the lysogenic cycle is adopted, the phage chromosome is
integrated (by covalent bonds) into the bacterial chromosome,
where it can remain dormant for thousands of generation
• The lytic cycle leads to the production of new phage particles
which are released by lysis of the host.

13. AGROBACTERIUM MEDIATED
TRANSFER
• Agrobacterium tumefaciens is a soil borne gram negative
bacterium.
• It invades many dicot plants when they are injured at the soil
level and causes crown gall disease.
• The ability to cause crown gall disease is associated with the
presence of the Ti (tumour inducing) plasmid within the
bacterial cell.
• Ti plasmid can be used to transport new genes into plant cells.

14. THE Ti-PLASMIDS
• A remarkable feature of the Ti plasmid is that, after infection, part of
the molecule is integrated into the plant chromosomal DNA .
• This segment, called the T-DNA, is between 15 and 30 kb in size,
depending on the strain.
• T-DNA contains eight or so genes that are expressed in the plant cell
and are responsible for the cancerous properties of the transformed
cells.
• These genes also direct synthesis of unusual compounds, called
opines, that the bacteria use as nutrient.

15. • The vir (virulence) region of the Ti- plasmid contains the
genes required for the T-DNA transfer process.
• The genes in this region encode the DNA processing
enzymes required for excision, transfer and integration of the
T-DNA segment.

16. • The T-DNA region of any Ti
plasmid is defined by the
presence of the right and the
left border sequences.
• These border sequences are 24
bp imperfect repeats.
• Any DNA between the borders
will be transferred in to the
genome of the plant.

17. Ti-Plasmid mediated
transfer of gene into a plant
• The Ti-Plasmid has an innate ability to transmit bacterial DNA
into plant cells.
• The gene of a donor organism can be introduced into the Ti
plasmid at the T-DNA region
• This plasmid now becomes a recombinant plasmid.
• By Agrobacterium infection, the donor genes can transferred
from the recombinant Ti- Plasmid and integrated into the
genotype of the host plant.

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VECTORLESS or DIRECT GENE
TRANSFER
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

19. Electroporation
• Electroporation uses electrical pulse to produce transient
pores in the plasma membrane thereby allowing DNA into
the cells.
• These pores are known as electropores.
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20. • 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.

21. Advantages of Electroporation
• 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

22. Microinjection
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

24. Limitations of microinjection
• Costly.
• Skilled personal required.
• More useful for animal cells.

25. Biolistics or Microprojectiles
• Biolistics or particle bombardment is a physical method that
uses accelerated microprojectiles to deliver DNA or other
molecules into intact tissues and cells.
• The gene gun is a device that literally fires DNA into target
cells .
• The DNA to be transformed into the cells is coated onto
microscopic beads made of either gold or tungsten.

26. • The coated beads are then attached to the end of the plastic
bullet and loaded into the firing chamber of the gene gun.
• An explosive force fires the bullet with DNA coated beads
towards the target cells that lie just beyond the end of the
barrel.
• Some of the beads pass through the cell wall into the
cytoplasm of the target cells

28. Liposome mediated gene transfer
• 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.
• Promote transport after fusing with the cell membrane.
• Cationic lipids are those having a positive charge are used for
the transfer of nucleic acid.

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

30. Calcium phosphate mediated DNA transfer
• The process of transfection involves the admixture of isolated
DNA (10-100ug) with solution of calcium chloride and
potassium phosphate so precipitate of calcium phosphate to be
formed.
• Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
• A fraction of cells will take up the calcium phosphate DNA
precipitate by endocytosis.

31. • Transfection efficiencies is quite low.

32. Polyethylene glycol mediated transfection
• This method is utilized for protoplast only.
• Polyethylene glycol stimulates endocytosis and therefore DNA
uptake occurs.
• Protoplasts are kept in the solution containing polyethylene
glycol (PEG).
• After transfer of DNA to the protoplast in presence of PEG
and other chemicals, PEG is allowed to get removed

33. SCREENING OF TRANSGENE
• The presence of transgene or gene of interest is detected by
several methods:
• A selectable marker gene
• Southern blot techniques
• Northern bolt technique
• Western blot technique

34. APPLICATION
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Clinical gene transfer applications
Vaccine Development
Production of transgenic animals
Treatment of Cancer, AIDS
Gene Discovery
Gene Therapy
Enhancing the resistance of plants
GMO

35. REFERENCES
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Sukharev, S.I., Klenchin, V.A., Serov, S.M., Chernomordik, L.V. & Chizmadzhev, Y.A. (1992). Electroporation
and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J., 63 (5):
1320-1327.
[2] Chu, G., Hayakawa, H. & Berg, P. (1987). Electroporation for the efficient transfection of mammalian cells with
DNA. Nucleic Acids Res., 15 (3): 1311-1326.
[3] Akamatsu, W., Okano, H.J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S.I., Miura, M., Matsuo, N.,
Darnell,
R.B. & Okano, H. (1999). Mammalian ELAV-like neuronal RNAbinding proteins HuB and HuC promote
neuronal development in both the central and the peripheral nervous systems. Proc. Natl. Acad. Sci. USA., 96
(17):
9885-9890.
[4] Osumi, N. & Inoue, T. (2001). Gene transfer into cultured mammalian embryos by electroporation. Methods., 24
Antoni Ivorra, Boris Rubinsky. "Gels with predetermined conductivity used in electroporation of tissue USPTO
Application #: 20080214986 - Class: 604 21 (USPTO)".
Jump upJump up^ Sugar, I.P.; Neumann, E. (1984). "Stochastic model for electric field-induced membrane pores
electroporation". Biophysical Chemistry 19 (3): 211–25. doi:10.1016/0301-4622(84)87003-9. PMID 6722274.
^ Alberts, Bruce; et al. (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN 978-0-81

36. •
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Jogdand, S.N. (2006). Gene Biotechnology. Himalaya Publishing House. Mumbai, India. 2nd ed., p 237-249.
Chen, C.A. & Okayama, H. (1988). Calcium phosphate-mediated gene transfer: a highly efficient trasfection
system for stably transforming cells with plasmid DNA. Biotechniques.,
Watwe, R.M. & Bellare, J.R. (1995). Manufacture of liposomes a review. Curr. Sci. India., . Nicolau, C., Legrand,
A. & Grosse, E. (1987). Liposomes as carriers for in vivo gene transfer and expression. Method Enzymol., 149:
157-176.
lies, M.A. & Balaban, A.T. (2001). Recent developments in cationic lipid-mediated gene delivery and gene therapy.
Expert Opin. Ther. Patents.,
Reece, R.J. (2004). Analysis of Genes and Genomes. John Wiley and Sons Ltd.
Johnston, S.A. & Tang, D.C. (1994). Gene gun transfection of animal cells and genetic immunization. Methods Cell
Biol., 43
Klein, T.M., Arentzen, R., Lewis, P.A. & Fitzpatrickmcelligott, S. (1992). Transformation of microbes, plants and animals by
particle bombardment. Biotechnology
King, R. (2004). Gene delivery to mammalian cells by microinjection. Methods Mol. Biol.,
David B. Burr; Matthew R. Allen (11 June 2013). Basic and Applied Bone Biology. Academic. p. 157. ISBN 978-0-12-391459-0.
Retrieved 15 July 2013.
^ Juan Carlos Lacal; Rosario Perona; James Feramisco (11 June 1999). Microinjection. Springer. p. 9. ISBN 978-3-7643-6019-1.
Retrieved 13 July 2013.