how gene are transferred chemically

1. GENE TRANSFER BY CHEMICAL
METHOD
BY- KHUSHI MANIKTALA
A005116520017
BTBM/20/113

2. GENE TRANSFER
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 integrated into host
genome
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.

3. TYPES OF GENE TRANSFER
DIRECT DNA TRANSFER
• PHYSICAL AND CHEMICAL METHODS
INDIRECT DNA TRANSFER
• AGROBACTERIUM AND VIRUS MEDIATED

4. CHEMICAL
METHODS • Calcium Phosphate
mediated gene
transfer
• Lipid mediated gene
transfer

5. Calcium Phosphate mediated gene transfer
It was the first chemical
transfection method to be used
with animal cells.
Calcium phosphate is probably
the most widely used transfection
method.
This is a simple, reliable method
applicable to many cultured cell
lines, and the reagents are
inexpensive.
It can be used both for transient
and stable transformation..

6. Historical Preview
The procedure was developed in 1973
by Graham and van der Erb for the
introduction of adenovirus DNA into
rat cells.
In a report by Szybalska and Szybalski
published in 1962 the presence of
calcium was shown to be responsible
for the successful transformation of
human cells with genomic DNA.
The first mammalian cell lines stably
transfected with plasmid DNA were
also produced by calcium phosphate
transfection, in 1978.

7. Principle
Calcium phosphate co-precipitation involves mixing DNA
with calcium chloride in a buffered saline/phosphate
solution to generate a calcium-phosphate-DNA co-
precipitate, which is then dispersed onto cultured cells.
Calcium phosphate facilitates the binding of the
condensed DNA in the co-precipitate to the cell surface,
and the DNA enters the cell by endocytosis.
Aeration of the phosphate buffer while adding the DNA-
calcium chloride solution helps to ensure that the
precipitate that forms is as fine as possible, which is
important because clumped DNA will not adhere to or
efficiently.

8. Methodology
Step 1: Mix DNA
Mix DNA with calcium chloride and add in a controlled
manner to a buffered saline/phosphate solution.
Step 2: Incubate
Incubate at room temperature to generate a precipitate of
extremely small, insoluble particles containing condensed
DNA.
Step 3: Add the DNA-calcium phosphate
Add the DNA-calcium phosphate co-precipitate to cells,
which adhere to the cell membrane. The co-precipitate
enters into the cytoplasm via endocytosis.
Step 4: Assay cells
Assay cells for transient gene expression or select for stable
transfection.

9. Advantages
 easily available
Inexpensive
Can be applied to wide
range of cell types Can be
used for transient and stable
transfection
Calcium phosphate appears
to provide protection against
intracellular and serum
nucleases.
Disadvantages:
low efficiency
It is not suited for in vivo
gene transfer to whole animals
Size and quality of the
precipitate are crucial to the
success of transfection
Toxicity, especially to primary
cellsits Sensitivity to slight
changes in pH, temperature
and buffer salt concentrations

10. Lipid Mediated Gene Transfer
Lipid Mediated Gene transfer method is also know as lipid
transfection.
 Method of transformation first described in 1965 as a model of
cellular membranes using liposomes.
Liposomes are artificial phospholipid vesicles used for the delivery.
 They can be preloaded with DNA by two common methods-
membrane-membrane fusion and endocytosis thus forming DNA-
liposome complex.
This complex fuses with the cell membrane of target cell and to
release the contents into the cell.
Animal cells, plant cells, bacteria, yeast protoplasts are susceptible to
lipofection method.

11. Liposome
Liposome are fluid filled spherical
vesicles made of phospholipids molecule.
Produced from glycolipids, cholesterols,
non-toxic surfactants and membranous
proteins.
Discovered in the year 1960 by British
hematologist Dr. Alec D. Bangham.
Manufactured and classified on the basis
of size, composition, charge and
speciality.
These liposomes work to deliver drug by
diffusion rather than by direct cell fusion.

12. Liposomes.
A schematic diagram of a stealth liposome con taining a
hydrophilic polymer (such as polyethylene glycol) to protect
it from destruction by immune cells, antibody molecules that
target it to specific body tissues, a water-soluble drug
enclosed in the fluid-filled interior chamber, and a lipid-
soluble drug in the bilayer.

13. Structure of Liposome
The walls of liposome consists of continous lipid bilayer organised
in same manner as that of the bilayer of natural membrane.
Membrane proteins can be inserted into liposomes (we can study
function of membrane protein in a much simpler enviornment).
Drugs or DNA can be linked to the wall of the liposomes or
contained at high concentration within its lumen.
Liposomes can protect from phagocytic cells of the immune system
by protective layer of synthetic polymer- polyethelene glycol.
The walls of the liposomes contains specific protein (hormones or
antibodies) that allow the liposomes selectively to the surface of
target cells.

14. Classification of Liposome
Liposomes are classified based on their structure as:
◦ Multi-laminar vesicles(MLV): made up of series of concentric bi-layer of lipid enclosing a small
internal volume.
◦ Oligolamelar vesicles(OLV): constitutes 2 to 10 bi layer of lipids surrounding a large internal volume
◦ Unilamellar vesicle(ULV): single layer of lipids
◦ Based on the size of the single layer they are further divide into the following types with in ULV as
1. Small unilaminar vesicle: size of 20 to 40 nm
2. Medium unilaminar vesicle: size of 40 to 80 nm
3. Large unilaminar vesicle: size of 100 to 1000 nm
4. Gaint unilaminar vesicle: size of more than 1000 nm

15. Action of Lipid Mediated Transfer
These are hollow microscopic spheres of
phospholipid, and can be filled with DNA or other
molecules during assembly. The liposomes will
merge with the membranes surrounding most
animal cells and the contents of the liposome end
up inside the cell, a process known as lipofection.
Although Lipofection works reasonably well, it is
rather nonspecific, because liposomes tend to
merge with the membranes of any cell.

16. Mechanism
Cationic lipids forming micellar structures
called liposomes are complexed with
DNA to create lipoplexes.
The structures fuse with the cell
membrane, at least sometimes after
interactions with surface proteoglycans.
The complexes are internalised by
endocytosis, resulting in the formation of
a double-layer inverted micellar vesicle.
During the maturation of the endosome
into a lysosome, the endosomal wall
might rupture, releasing the contained
DNA into the cytoplasm and potentially
towards the nucleus.
DNA imported into the nucleus might
result in gene expression. Alternatively,
DNA might be degraded within the
lysosome

17. ADVANTAGES
Economic
 Efficient delivery of nucleic
acids to cells in a culture dish.
Delivery of the nucleic acids with
minimal toxicity.
Protection of nucleic acids from
degradation.
Easy to use, requirement of
minimal steps and adaptable to
high-throughput systems.
DISADVANTAGES
It is not applicable to all
cell types.
It fails for the transfection
of some cell lines with
lipids.
Low efficiency in-vivo.

18. Application
Gene therapy, in which a therapeutic gene is transferred to cells,
is potentially promising and various gene transfer systems have
been developed and tried experimentally and clinically.
Currently, in vivo gene transfer with expression plasmid vector
is one of the emerging remedies because gene transfer with
plasmid vector is simple and clinically safe compared to the
other transfer systems with virus vectors. However, large
amounts of plasmid vectors have been used in these studies,
suggesting the low efficiency of gene transfer.
On the other hand, calcium phosphate (CaP) precipitate, in
which plasmid vector is incorporated, has been used for in vitro
gene transfer
Since CaP precipitate stabilizes nucleic acid it is speculated that
CaP precipitate would be also useful for in vivo gene transfer.

19. Conclusion
Thus there are a number of ways by which the gene can be
introduced into the cells.
With the advent of molecular tools and technologies it is
now comparatively easy to introduce gene into cells
without loosing its integrity and biological activity.
Moreover the recent development in molecular biology has
made the transfer of gene with great accuracy to the target
cell.
The transfer of gene through different gene transfer
technologies has cured a number of diseases.
Research is on progress to cure those diseases which
cannot be cured by using drugs.
Moreover the treatment of diseases by gene transfer
provides better result for a prolong period of time. It is the
need of hour to discover new and cheap method of gene
transfer technologies so to make the treatment of the
diseases a little easier and affordable.