Genetic

Transformation

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Lecture- 35

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•The gene cloning method described by Stanley Cohen and coworkers

requires the introduction of the recombinant vector molecules into

suitable host cells. These researchers used transformation method to

introduce the recombinant plasmid molecules into E. coli cells.

•Transformation is the entry or introduction of naked DNA into the living

cells. In the year 1944 Oswald Avery, Colin MacLeod and Maclyn

McCarty proved that the genetic transformation discovered by

Frederick Griffith in 1928 in Diplococcus pneumoniae was due to the

introduction of naked DNA into the bacterial cells.

•The transfection method developed by Morton Mandel and Akiko Higa

(1970) for E. coli became the basis for the subsequent bacterial

transformation methods. The transformation of E. coli with the purified

plasmid DNA was achieved for the first time by Stanley Cohen and

coworkers in the year 1972.

INTRODUCTION

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Subsequently, the following organisms/cells of

organisms were transformed for the first time in the

years mentioned in brackets: yeast (1978), cultured

mammalian cells (1980), Drosophila melanogaster

(1982) and cultured plant cells (1983).

In 1987, B. M. Chassy and J. L. Flickinger

introduced the electroporation technique for

transformation of living cells. In the same year,

Theodore Klein, Edward Wolf, Ray Wu and John

Sanford developed high velocity microprojectiles to

deliver DNA into cells.

On the basis of this method a gene gun machine for

commercial use was developed by DuPont company.

Genetic transformation is the entry or

introduction of naked DNA into the living

cells.

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GENETIC TRANSFORMATION METHODS

Stimulation by chemicals

Encapsulation in liposomes

Electroporation

Microprojectile bombardment

Microinjection

Cell perforation using SiC whiskers

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STIMULATION BY CHEMICALS

1970: E. coli was transformed by calcium

chloride and heat shock treatments

1977: Lurquin and Kado demonstrated that

uptake of plasmid DNA by cowpea

protoplasts was stimulated by poly-L-

ornithine (PLO).

PLO, a polycation, possibly stimulates the

uptake of nucleic acids in plant protoplasts

by neutralising their surface charge.

PEG in the presence of calcium ions (CaCl2)

is used for transforming plant protoplasts.

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The technique involves incubating

protoplasts with naked DNA in the presence

of PEG and CaCl2

After gradual dilution of mixture, protoplasts

are collected, washed and finally plated on

selective medium.

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ENCAPSULATION IN LIPOSOMES

Liposomes are microscopic lipid molecules

which are produced when phospholipids are

dispersed in an aqueous phase.

Liposomes are large enough to allow

entrapment of DNA and can interact with a

variety of cells. Negatively charged

unilamellar liposomes are best suited for the

transfer of DNA and RNA into plant cells.

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ELECTROPORATION

The process by which macromolecules

present in the extra cellular medium are

internalized by living cells on exposure to

a brief electric pulse.

The technique allows introduction of DNA

into living cells/protoplasts by passing

short pulse of electricity which has a

voltage peak value of 250-350 volts with

RC constant in milliseconds range.

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MICROPROJECTILE BOMBARDMENT

Exogenous DNA molecules are adsorbed on

the surface of microscopic tungsten

microprojectiles which are then accelerated

to high velocities by a particle gun onto the

target cells.

The advantage in this approach is that a

naked DNA molecule can be directly

transferred into intact living cells or tissues.

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Particle gun for accelerating microprojectiles

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MICROINJECTION

Living cells/protoplasts are surface attached

on a slide by embedding in agro or polylysine

using a holding pipette.

The DNA is transferred by injecting it with

micro needles into the surface attached

cells/protoplasts.

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Microinjection by the holding pipette method13

CELL PERFORATION USING SiC WHISKERS

Recipient cells are perforated by vortexing in

the presence of SiC (silicon carbide)

whiskers along with DNA.

Cell perforation thereby allows direct entry of

exogenous DNA.

This method was initially used for genetic

transformation of maize suspension cells and

later extended to transform other plants.

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