Pulmonary drug delivery system M.pharm -2nd sem P'ceutics
Methods of gene transfer.
1. RVSKVV
COLLEGE OF AGRICULTURE ,INDORE
DEPT- GENETICS & PLANT BREEDING
SUBJECT- Biotechnology for crop
improvement.
SUBMITTED TO- SUBMITTED
BY-
Dr.Kanak Saxena Charu Jamnotia
2. Gene transfer- Introduction
Gene transfer is the mechanism by which
DNA is transferred from a donor to a recipient.
Once donor DNA is inside the recipient,
crossing over can occur.
The result is a recombinant cell that has a
genome different from either the donor or the
recipient.
3. Artificial methods of DNA
transfer-
PHYSICAL METHODS-
1) Macroinjection
2) Microinjection
3) Protoplast fusion
4) Biolistics transformation
4. Chemical methods-
1. DNA transfer by Calcium Phosphate
method.
2. Transfer of DNA by use of polyeythene
glycol.
3. Use of DEAE- Dextran for DNA transfer.
4. Liposome mediated transfer.
6. GENE TRANSFER
TECHNOLOGIES
Gene transfer technology provides the ability
to genetically manipulate the cells of higher
animals.
During the 1970s it became possible to
introduce exogenous DNA constructs into
higher eukaryotic cells in vitro.
Mammalian transgenesis was first achieved in
1980s.
7. Microinjection -
o DNA can be introduced into cells or
protoplast with the help of very fine needles or
glass micropipettes of diameter 0.5 to 10 µm.
8. Gene Gun -
Simplest method of direct introduction of
therapeutic DNA into target cells.
Looks like a pistol but works like a shotgun
Golden pellets.
First discribed by John Sanford at Carnell
University in 1987.
9.
10. DNA become sticky
adheres to biologically
inert particles such as
metal atoms (gold or
tungustun)
Accelerating this DNA
particle complex in a
particle vaccum and
placing the target tissue
within the accelerating
path gathers the DNA.
Cells that take up the
desired DNA,identified
through the use of a
marker gene or then
cultured to replicate the
gene & possibly cloned.
11. Calcium Phosphate Method-
o The process of transfection involves the admixture of
isolated DNA (10-100 µg) with solution of calcium
chloride and potassium phosphate under condition
which allow the precipitate of calcium phosphate to be
formed.
o Cells are then incubated with precipitated DNA either in
solution or in tissue culture dish.
o A fraction of cells will take up the calcium phosphate
DNA precipitate by endocytosis.
12.
13. Polyethlene glycol mediated
transfer-
This method is utilised for protoplast only.
Polyethylene glycol stimulates endocytosis
and therefore DNA uptake occurs.
Protoplast are kept in the solution contaning
polyethylene glycol (PEG).
After transfer of DNA to the protoplast in
presence of PEG and other chemical, PEG is
allowed to get removed.
14.
15. 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 transfer of nucleic acid.
16.
17. Electroporation-
o It uses electrical pulse to produce transient pores
in the plasma membrane thereby allowing
macromolecules into the cells.
o Microscopic pores are induced in biological
membrane by the application of electric field.
o These pores are known as electropores which allo
molecules,ions & water to pass from one side of
the membrane to another.
