CHAPTER 5 :

TRANSGENESIS
Gene Transfer into Eukaryotic
Cells & Mammalian Embryos

At the end of the lecture, students

should be able to:
Define transgenesis
Describe gene transfer

method
Explain the transgenesis
processes

Transgenesis
the process of
introducing a foreign gene
(transgene) into aliving
organismso that the organism
will exhibit a new property and
transmit that property to
itsoffspring.

Transgenesisis

Methods of Gene Transfer

There are three types of genetic material, double

stranded gene segments, oligonucleotides and
cDNA sequences.
Transfection is the method of introducing new
DNA into a eukaryotic cell.
Physical
methods
Microinjection
Biolistic
Electroporati
on

Chemical
methods
Lipids and
liposome
mediated
gene transfer
Non
liposomal

Biological
methods
Conjugation
Transformation
Transduction
Agrobacterium
mediated
transfer

Methods of Gene Transfer

transfer can be transient or
stable
In transient transfection, DNA is not
integrated to the host chromosome
(temporary but high expression of target
gene)
In stable transfection, the foreign DNA
is integrated into the chromosomal
DNA and the genetics of the recipient
cell will be forever changed
Gene

Microinjection
Microinjectionis

the use of a
glassmicropipetteto inject a liquid
substance into a living cell but may also
include intercellular space.
Microinjection usually involved aninverted
microscopewith amagnification powerof
around 200x.
Sometimes it is performed using a
dissectingstereo microscopeat 40-50x or a
traditionalcompound upright microscope.

Microinjection

To transfer genes into animals;

Inject the rDNA with a fine glass needle into the
pronuclei present in the embryo, 12 14
hours after fertilization
Apronuclei is the nucleus of a sperm or an egg
cell during the process of fertilization, after the
sperm enters the ovum, but before they fuse.
Sperm and egg cells are haploid, meaning they
carry half the number of chromosomes.
Transfer the embryo to a recipient mother
where it can grow and develop into a normal
animal

Biolistic
Microparticle

gun / Agene gunor

abiolistic or bioballistics particle
delivery
system

particle
bombardment
where
accelerated
microprojectiles
deliver
DNA
straight into cells
DNA to be transformed are coated into
microscopic beads coated with
gold/tungsten

Biolistic
The coated beads are
attached to the end of
the plastic bullet and
loaded into the firing
chamber of the gene
gun.
An explosive force fires
the bullet with DNA
coated beads towards
the target cells that lie
just beyond the end of
the barrel.
Some of the beads
pass through the cell
wall into the cytoplasm
of the target cells

Electroporation
Electroporation

DNA can be
delivered directly into cells using an
electrical pulse.
Electroporation caused anelectrical
fieldto be applied to cells in order to
increase the permeability of thecell
membrane (pore formation).
This
allowed
chemicals,
drugs,
orDNAto be introduced into the cell.

Electroporation

Chemical
methods
Liposomes
are spheres
of lipids
that can
transport
molecules
into cells
Promote
transport
after fusing
with cells.

Calcium phosphate mediated

gene transfer (Non-liposomal)
Isolated

DNA were mixed with a

mixture of Calcium chloride and
Potassium phosphate.
The solution were incubated with the
cells
A fraction of the cells will take the
calcium phosphate precipitate via
endocytosis.

Calcium phosphate
mediated gene transfer

Biological method of gene transfer

Widely used strategy to get genes into
eukaryotic cells, leading to transcription &
translation.
Four methods
i.
Conjugation
ii. Bacterial transformation
iii. Retroviral transduction
iv. Agrobacterium mediated transfer

Conjugation

Transformation
Direct

uptake of exogenous DNA through

the cell membrane by a competent cell.

Viral-mediated gene transfer

Transductionis

the process by which

DNA
is
transferred
from
one
bacterium to another by a virus.
It also refers to the process whereby
foreign DNA is introduced into
another cell via a viral vector.

Agrobacterium mediated
transfer
Agrobacterium tumefaciens is a soil borne
gram negative bacterium.
It invades many dicot plants when they are
injured at the soil level and causes crown
gall disease.
The ability to cause crown gall disease is
associated with the presence of the Ti
(tumour inducing) plasmid within the
bacterial cell.
Ti plasmid can be used to transport new
genes into plant cells.

Ti Plasmid Infection
infects plant, transfers Ti
plasmid to host cells
T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
Bacterium

Ti Plasmid Infection

Transgenic

Transgenic Animals
Ralph Brinster & Richard Palmiter, 1981 introduced gene for rat growth hormone
(GH) into fertilized mouse eggs
Injected DNA constructed to have rat GH
gene coding region just downstream from
mouse metallothionein gene promoter (a
strong promoter)
Treat mice with metals or glucocorticoids
> GH induced, mice got very big

Transgenic Animals
Transgenic animals useful for determining
effects of overexpression of particular
DNA sequence
Transgenic animals also being developed as
part of agricultural biotechnology
Place foreign growth hormone genes into
cow > grow much leaner than controls
lacking gene
Excess
growth
hormone
stimulates
conversion of nutrients into protein rather
than fat (leaner)

Transgenic plants
Introduce

gene into cultured plant cells

> grow into mature plants with
foreign gene
During
infection, Ti plasmid of
bacterium enters plant cell &
incorporates
into
plant
chromosomes
Induces cell to proliferate & provide
nutrients for bacteria
Ti plasmid can be isolated from
bacteria & linked with foreign genes
> make recombinant plasmid

Use of the T-DNA Plasmid

The benefit
to introduce bacterial
genes for insect-killing toxins;
protect
plants
from
insect
predators
Significant
increase
in
photosynthetic efficiency >
great
increases
in
crop
production
Used

The benefit
Bacterial

genes involved in nitrogen

fixation activity & introduce them into
chromosomes
of
nonleguminous
plants that now depend on added
fertilizer for reduced nitrogenous
compounds
Identify genes responsible for N2
fixation in Rhizobium > deliver to
plants > less fertilizer

Therapeutic Proteins/Antibodies

Therapeutic

proteins are proteins that

are engineered in the laboratory for
pharmaceutical use.
Insulin was the first therapeutic
protein to be introduced to treat
diabetes in the 1920s.
The
fastest
growing
class
of
therapeutic proteins is antibodies.