GENE TRANSFER TECHNOLOGY

Presented By
KRISHNAKUMAR M S
Reg No: 1421316026
II-M.Sc., Bio Chemistry
Government Arts College - Pmk
INTRODUCTION
 Gene transfer is to transfer a gene from one DNA molecule to
another DNA molecule
 The efficiency of this process is often crucial for determining the
success of cloning.
 The transferred gene is known as a transgene and the organism
that develop after a successful gene trasnfer is known as
transgenic
 DNA is hydrophilic molecule and it can not pass through the
plasma membrane
 METHODS OF GENE TRANSFER:
 VECTOR LESS GENE TRANSFER:
A. Electroporation
B. Lipofection
C. Microinjection Direct Transfer
D. Particle Bombardment
 VECTOR MEDIATED GENE TRANSFER:
A. Transduction
B. Transformation
C. Conjugation
VECTOR LESS GENE
TRANSFER
ELECTROPORATION
PRINCIPLE:  Apply to the high electric impulses
 Use of high electric field
 Formation of pores
 Permeabilize the cell membrane
 Uptake of DNA  DNA enters and get integrated into

PROCEDURE: hose cell

 Pores seals with the foreign
 Sample is incubated in
DNA inside
buffer with foreign DNA
LIPOSOME MEDIATED GENE TRANSFER
 Liposomes are artificially created lipid vesicles

 It is like phospholipid membrane

 Successfully used in mammalian cells

 Used in Drug delivery

 The efficiency of transformation increases with Use of PEG-

poly ethylene glycol.

MECHANISM:
 Adhesion of liposomes to
surface
 Fusion at the site of
attachment
 Release of plasmid into the
cell
DIRECT TRANSFER OF DNA:

 It is possible to directly transfer the DNA into the cell

nucles
 There are two techniques are commonly used for this
purpose
1. Microinjection
2. Particle Bombardment
MICROINJECTION:
 The rDNA is directly injected into the nucleus of host
cells
PARTICLE BOMBARDMENT
 Microprojectile bombardment is a most effective physical

gene transfer method

 Also called a biolistics (biological+ballistics)

 Other names are Particle gun, Gene gun, Bio blaster

 Widely used particle bombardment apparatus PDS-1000/HS.

PARTS:
1. Rupture Disc
2. Macro carrier
3. Micro carrier(vector
DNA Coated)
4. Stopping plate
5. Sample
Mechanism:
 Micro carriers ( gold particles coated with DNA) carried by Macro carriers

 Macro carriers are inserted and pushed downward by rupturing the disc.

 The stopping plates does not permit the movement of macro carriers.

 But microbe carriers are propelled at a high speed into the sample

 DNA are released which integrate with the genome.

VECTOR MEDIATED GENE
TRANSFER
TRANSDUCTION
 The process of transfer of DNA into a host cell via virus particle.

 This process known as transduction

 Which vector is used in transduction process they are called

transducing vector
 The bacteriophage vector or transducing phage are used in this

method
There are two type of transduction,

Generalized transduction
Specialized transduction
GENERALIZED TRANSDUCTION:
1. Penetration:
 The phage injects the phage DNA into the cytoplasm of the bacterium
(donor)
2. Fragmentation:
 During the lytic cycle of viral replication the phage DNA, along
with the bacterial DNA is browken down into smaller pieces
3. Maturation and packaging:
 Some part of the bacterial chromosome is then packaged into one
of the viral capsids when that is released by lysis of the bacterium.
4. Transfer:
 The transducing phase with the bacterial chromosome now

infects a second bacterium (recipient bacterium), and the donor

DNA enters the cytoplasm of the second bacterium.
5. Integration:
 Donor DNA integrates with the recipient bacterium by
homologous recombination
Results in Generalized Transduction:
There are two types of result can obtain,

1. Abortive Transduction
Transient Expression of the transferred gene
2. Completed Transduction
Inherited to Next generation.
SPECIALIZED TRANSDUCTION:
1. Penetration:
 Phage DNA injected into the bacterium
2. Integration:
 the phage DNA is integrated into the bacterial
chromosome during the lysogenic cycle.
3. Restriction:
 Due to the imprecise cutting of the phage DNA, some
part of the bacterial chromosome is also excised.
4. Synthesis and Transfer:
 The phage containing some part of the bacterial
chromosome then infects a new host, and the donor DNA is
incorporated into the recipient bacterium during the
lysogenic cycle of the replication.
5. Expression:
 The recipient then expresses the newly acquired genetic
trait.
TRANSFORMATION:
 Treatment of host cells with divalent calcium
 Increase the chances of DNA entry to host
 Incubation of cells on ice
 Add rDNA
 Again transferred in ice
 After these are placed at 37-45°C
 This process enable the bacteria to takeup DNA
 Some time CaCl2 may result inn precipitate and toxicity to the cell so
some workers use DEAE-D for DNA transfer
CONJUGATION:
 Conjugation is the transfer of a plasmid or other self-transmissible DNA

element and sometimes chromosomal DNA from a donor cell to a recipient

cell via direct contact usually mediated by a conjugation pilus or sex pilus.
 Recipients of the DNA transferred by conjugation are called

transconjugants.
 Plasmid: Extra chromosomal DNA present in inside of the cell, they can

replicate independently.
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