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
• 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).
8.
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.
10.
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.
12.
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.
18. 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.
•
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
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
27.
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
• 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.
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
• 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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