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GENE DELIVERY SYSTEMS
— Gene is a segment of DNA that codes RNA for polypeptide molecule
...

— Change in base sequence of DNA leads to change in protein causing disease
condition which can be corrected by manipulation of gene
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TRANSFORMATION: It is the uptake of DNA by whole cells from the
surroundings
...

BACTERIAL TRANSFORMATION- HISTORICAL ACCOUNT
·
·

First demonstrated by Griffith in 1928 in the TRANSFORMING
PRINCIPLE experiment
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MECHANISM FOR TRANSFORMATION

·

·

·

·

Long ds molecule bind to the surface of the cell with the
help of DNA-binding protein
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The strand which is not degraded associates with
competence specific protein
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COMPETENCE:
Transformation has two crucial steps:
1
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2
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Bacteria can be naturally competent or competency can be artificially
induced in bacteria with the help of certain chemicals such as calcium
chloride
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The transformation frequency of very competent cells is around 10-3
even when excess of DNA is used
...
Competence appears to be
associated with the synthesis of cell wall material at a particular stage of
life cycle
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The other methods of transformation include:

·

·

·

Physical methods like microinjection
Electrical methods like electroporation
Liposome-mediated transfer of DNA
CALCIUM CHLORIDE METHOD:
1
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2
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3
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CaCl2
causes the DNA to precipitate on to the outside of
the cell or perhaps induces a change in the cell wall
that improves DNA-binding
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Heat shock for two minutes at 42o C stimulates the
DNA movement from cell exterior into the cytoplasm
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Transformed cells are incubated in the nutrient
broth at 37oC for 60-90 minutes
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NOTE 1- Rubidium chloride can replace calcium
chloride
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ADVANTAGES
1
...
Success rate is 0
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DISADVANTAGE
This process becomes critical in cloning experiment
when a large number of recombinants are required
or when starting material is limited
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However, if the recipient carries a homologous plasmid
and if the same restriction cut occurs within the homologous marker, then same
marker transforms efficiently
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TRANSFECTION:
It is equivalent to transformation
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In this, purified phage DNA or recombinant phage
molecule is mixed with competent cells and the DNA uptake is induced by heat
shock
...
coli cells
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Plant cells pose a
problem of a rigid cell wall which is a barrier for DNA uptake
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Protoplasts take up the DNA readily
...
microinjection and biolistics
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Various modifications have been made to increase the transfection efficiency
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Formation of calcium phosphate- DNA complex in the test tube by mixing
calcium chloride and HEPES buffer of pH 7
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The precipitate is then added to culture dishes containing rapidly
growing cells
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2
...
95
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Instead, after addition of mixture to the culture
dishes, the precipitate is slowly formed before it is actively taken in by the
cells
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ADVANTAGES:
1
...

2
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DISADVANTAGES:
1
...

2
...

3
...

4
...

5
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NOTE: Other chemical transfection methods involve the use of polycationic
carbohydrate- Diethylaminoethyl dextran (DEAE- dextran) or polycationic
detergent polybrene
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— This is polycationic, high molecular weight substance and convenient for
transient assays in cos cells
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— If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO)
shock, then upto 80% transformed cell can express the transferred gene
...
Treatment with
chloroquinone increases transient expression of DNA
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TRANSFORMATION OF PROTOPLASTS
Direct DNA uptake by protoplast is stimulated by chemicals like
polyethyleneglycol (PEG)
...
This is followed by cell wall
formation and initiation of cell divison
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OR

— This method is utilized for protoplast only
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— Protoplasts are kept in the solution containing polyethylene glycol (PEG)
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— Calcium chloride is added and sucrose and glucose act as osmotic buffering
agent
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— Intact surviving protoplasts are then cultured to form cells with walls and
colonies in turn
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— PEG based vehicles were less toxic and more resistant to nonspecific
protein adsorption making them an attractive alternative for nonviral gene
delivery
...
It was initially applied to animal cells and has subsequently been
successful with plant cells
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5 micrometer diameter) is made
I
Cooled and fitted on microinjection apparatus

I
Process now can be carried out with great precision while viewing through
ocular lens
...

I
Cells to be microinjected held by little suction through ordinary glass
pipette or immobilized on solid support
I
Microneedle is guided to the target cell, tip inserted, contents emptied and
needle withdrawn
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APPLICATIONS:
— Process is applicable for plant cell as well as animal cell but more common
for animal cells
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— Procedure is important for gene transfer to embryonic cells
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— Method has been successfully used with cells and protoplast of tobacco,
alfalfa etc
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2
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4
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Chances of DNA integrates to a cell is high
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Effective in transforming primary cells as well as cells in established
culture
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DNA injected in this process is subjected to less extensive modifications
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Mere precise integration of recombinant gene in limited copy number
can be obtained
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Requires complex specialized equipment
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Skilled operator needed
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A good specialist can only inject approx
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4
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5
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6
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7
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8
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9
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BIOLISTICS (PARTICLE GUN METHOD OR GENE GUN)

1
...
It involves
shooting the DNA into the cells
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Tungsten or gold microparticles coated with DNA known as
MICROPROJECTILES are accelerated to a very high velocity (1400 ft
...

3
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4
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5
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These microprojectiles carry the DNA into the cell
and in some cases, stable transformation occurs
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— Genomes of subcellular organelles have been accessible to genetic
manipulation by biolistic method
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— Method can be applied to filamentous fungi and yeast (mitochondria)
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ADVANTAGES:
— Thousands of particles are accelerated at the same time resulting in the
transfer of genes into many cells simultaneously
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— Requirement of protoplast can be avoided
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— Manipulations of the genomes of the sub-cellular organelles can be
achieved
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2
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4
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Suitable only for monolayers
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LIPOSOME- MEDIATED DNA TRANSFER
1
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2
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3
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4
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STEPS:
— Liposomes for use as gene transfer vehicles are prepared by adding an
appropriate mix of bilayer constituents to an aqueous solution of DNA
molecules
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— This self-organizing process creates discrete spheres of continuous lipid
bilayer membrane enveloping a small quantity of DNA solution
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ADVANTAGES:
1
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2
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3
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4
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5
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DISADVANTAGES:
1
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2
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