Direct Gene Transfer Methods

1. -Saugat
Bhattacharjee
Dept. of Genetics

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Genetic 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.

6. GENE TRANSFER TECHNOLOGIES
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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 (germline) transgenesis was first
achieved in the early 1980s.
The model used in this study was mice.

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DNA can be introduced into cells or protoplast with the
help of very fine needles or glass micropipettes having
the diameter of 0.5 to 10 µm.
Computerized control of holding pipette, needle,
microscope stage and video technology has improved
the efficiency of this technique.

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Simplest method of direct introduction of therapeutic DNA into target
cells
Looks like a pistol but works more like a shotgun Golden Pellets.
First described as a method of gene transfer into plants John Sanford
at Cornell University in 1987.
This device is able to transform almost any type of cell, including
plants, and is not limited to genetic material of the nucleus: it can
also transform organelles, including plastids.

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DNA (or RNA) become “sticky,”
adheres to biologically inert
particles such as metal atoms
(usually tungsten or gold)
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accelerating this DNA-particle
complex in a partial vacuum and
placing the target tissue within
the acceleration path gathers the
DNA
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cells that take up the desired
DNA, identified through the use
of a marker gene are then
cultured to replicate the gene
and possibly cloned
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most useful for inserting genes
into plant cells such as pesticide
or herbicide resistance

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The process of transfection involves the admixture of
isolated DNA (10-100ug) with solution of calcium
chloride and potassium phosphate under condition
which allow the 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.

15. Limitations of calcium phosphate mediated DNA
transfer:
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Frequency is very low.
Integrated genes undergo substantial modification.
Many cells do not like having the solid precipitate
adhering to them and the surface of their culture
vessel.
Integration with host cell chromosome is random.
Due to above limitations transfection applied to
somatic gene therapy is limited

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It uses electrical pulse to produce transient pores in the
plasma membrane thereby allowing macromolecules
into the cells.
Microscopic pores are induced in biological membrane
by the application of electric field.
These pores are known as electropores which allow the
molecules, ions and water to pass from one side of the
membrane to another.

18. 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.