Transgenic Animals

A genetically modified organism (GMO) is an

organism whose genetic material has been altered
using techniques in genetics, generally known as
recombinant DNA technology.
Recombinant DNA technology is the ability to
combine DNA molecules from different sources into
the one molecule.
Thus, the abilities or the phenotype of the
organism, or the proteins it produces, can be
altered through the modification of its genes.
Transgenics are genetically modified organisms
with DNA from another source inserted into their
genome.
A large number of transgenic animals have been
created

Production of Transgenic Animals

Transgenic animals can be produced by three
different techniques:
Method 1.
Microinjection of DNA into the
pronucleus of zygotes
Method 2.
Injection of embryonic stem cells
into blastocysts.
Method 3.

Use of engineered retroviruses

The procedure is done with the help of a

complete microinjection set-containing of a

A pronucleus (plural: pronuclei) 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.

Method 1: Microinjection of DNA into

the pronucleus

The
pronuclear
microinjection
method
of
producing
transgenic animals is based on the introduction of linear DNA
sequences into the chromosomes of fertilized eggs.
The foreign DNA must be integrated into the genome prior to
the doubling of the genetic material that precedes the first
cleavage in order for the animal to be born with a copy of
this new information in every cell.
For several hours following the entry of the sperm into the
oocyte, the male and the female pronuclei can still be seen
individually under a normal light microscope and they have
not fused yet into a so called zygote.
The foreign DNA may be injected into either pronuclei with
no difference in results; however, the DNA is typically
injected into the male pronucleus because it is slightly larger
and closer to the oocyte surface.
These oocytes are subsequently transferred into the uterus
of pseudo-pregnant recipient animals. The offspring is

Creation of Transgenic Animals by Nuclear

Injection

In vitro fertilization is used to

start a transgenic animal.

Harvested eggs and sperm are

fertilized, and before the
pronuclei fuse, the transgene is
injected into the male
pronucleus.

The embryo continues to divide

in culture and is then implanted
into a mouse.

The foster mother mouse has

been treated with hormones so
that she accepts the embryo
and carries on with the
pregnancy.

The offspring are screened for

stable integration of the
transgene.

Gene injected into

the male pronuclei

Method 2: Injection of ES cells into

blastocyst-stage embryos
Embryonic stem cells (ES cells) are derived from the inner
cell mass of blastocysts (early embryos). These cells are
pluripotent, which means that they can develop into almost
any type of tissue.
ES cells are used for more precise modifications of the
mouse genome. This technique makes it possible to insert
as well as remove or modify DNA sequences. Knock-out,
and conditional mutant mice can be produced with this
method.
The first step is the removal of ES cells from a blastocyst.
After transfection of the ES cells; selection, cloning and
screening methods make it possible to detect ES cell clones
that demonstrate the desired, site-specific recombination.
After microinjection of the genetically modified ES cells into
blastocyst-stage embryos, the ES cells divide and become

To create a transgenic animal with embryonic stem cells, the

transgene must first be inserted into the embryonic stem
cells. The stem cells are transformed with the transgene,
which integrates by homologous recombination.
Then the stem cells are injected into an early male embryo
(blastocyst) of mouse and the embryo is put into a pseudopregnant female mouse. The offspring are chimeras because

Method 3: Use of engineered retroviruses.

Retroviruses can infect the cells of early embryos,
including embryonic stem cells. Hence, retrovirus
vectors may introduce transgenes.
Disadvantages
Virus DNA is introduced along with the transgene.
Retroviruses can carry only limited amounts of DNA.
Founder animals made using retroviruses are
always chimeras, because insertion of the virus does
not occur exactly when the nuclei fuse.
Partially transgenic animals (that have some sectors
or tissues altered) are useful because the transgenic
tissues may be compared with normal tissue within
the same animal.

Goals
of
creation

transgenic

animal

1. Improve livestock animals

2. Research into animal and human
disease
Usetransgenic
of animals as
bioreactors
The3.first
animals
were mice created by
Rudolf Jaenisch in 1974. Jaenish successfully managed
to
insert
foreign
DNA
into
the
early-stage
mouse embryos; the resulting mice carried the
modified gene in all their tissues.
There has also been the genetically manipulated bull.
A human gene (responsible for production of
lactoferrine) was inserted into bulls genetic code
while in an early embryonic stage in 1990. As a result,
milk from his female descendants contained the
human protein lactoferrine, which can be used as
medicine, but it was present at such low levels that it
was not profitable to extract them.

Large Transgenic Mice illustrate transgenic

technology
Creation of larger mice by inserting the rat gene for
growth hormone (Somatotropin)
Somatotropin consists of a single polypeptide encoded
by a single gene. Somatotropin gene of rat was cloned
and inserted into fertilized mouse eggs. The transgenic
mice were larger (about twice normal size), although
not as large as rats.
The gene was under the control of the promoter
from an unrelated mouse gene, metallothionein ,
which is normally expressed in the liver.
Instead of being made in the pituitary gland (the
normal
site
for
growth
hormone)
the
rat
somatotropin was manufactured in liver
Human somatotropin has also been expressed in mice

Large Transgenic Mice

Trendy Transgenic Mice

Marathon Mouse
Can run about 1800 metersmore than a milebefore
exhaustion. This is twice as far as a normal mouse can last.
Marathon mouse has enhanced PPAR-deltaa regulator of
several genes involved in burning fat and in muscle
development.
Mighty Mouse
Lack myostatin, a protein that slows muscle growth. The
result is colossal muscle development.
Fierce Mouse
Shows abnormal aggression. Both copies of the NR2E1 gene
deleted.
The cloned human version of the NR2E1 gene has been shown
to cure the defects in brain development and restore normal
behavior to fierce mice.
Smart Mouse
Having improved learning and memory.
Has extra copies of the NR2B gene encoding the NMDA

Recombinant protein production using

transgenic livestock
Production of heterologous proteins

Recombinant proteins can be produced in the milk from

transgenic cows or other farm animals.
The cloned genes are placed under the control of a regulatory
region that will allow gene expression only in the mammary
gland.

For small-scale production, transgenic goats are often

used.
E.g. transgenic goats producing rTPA (recombinant tissue
plasminogen activator)

Knockout mice for medical research

Transgenic animals, mostly mice, are of great value in
the genetic analysis of inherited diseases and cancer.
The general approach is to inactivate, or knock out,
the gene of interest and then ask what defect this
causes.
The gene is inactivated first and then this copy is used
to generate knockout.
The incoming DNA, carrying the disrupted gene,
replaces the original & functional copy of the gene by
homologous recombination.
Founder mice are obtained that have one copy of the
disrupted gene. By breeding them together, mice with
both copies disrupted are obtained.
If the gene in question is essential, homozygous

Knockout Mice
The target gene is cloned and
disrupted by inserting a DNA
cassette.
This work is usually done in
bacteria.
Once the construct is made,
it is put
back
into
a
mouse
by
injection into
the male pronucleus during
fertilization.
After the transgenic offspring
are born, two heterozygotes
are crossed to create a
homozygous
knockout
mouse.

Location effects on expression of the transgene

Transgenic organisms carrying the same inserted
transgene often differ considerably in expression.
Both the level of expression and the pattern of expression
in various
tissues of the body may vary.
Many of these effects are due to the location of the
transgene.
Presence of any nearby regulatory elements like
enhancers.
The physical state of the DNA is important.
Determining the position effect
Extract transgene DNA from a transgenic animal in
which the
transgene is not expressed.
Insert the DNA
transgenic animals.

to

construct

another

line

of

Combating location effects on transgene

expression
A. Building appropriate regulatory elements in the
transgene construct itself.
B. Site-directed insertion

1. Dominant control elements

Some regulatory sequences control nearby genes or clusters of
genes in a dominant manner.

2. Insulator sequences or boundary elements

These sequences block the activity of other regulatory
elements.
If a gene is flanked by two insulator sequences it is protected
from the effects of any regulatory elements beyond the
insulators.
Transgenes flanked by insulators probably form independent
loops of DNA from
which heterochromatin is excluded.

Protection of a Gene by Insulator Sequenc

Targeting the transgene to a specific location

Requires homologous recombination, as opposed

to the random integration that usually happens
with injected DNA.

Inserting a transgene in a specific location may

be desirable for several reasons.
1. Chromosomal location often affects the
expression of a transgene.
2. The transgene is not necessarily a novel
gene. Gene replacement in the same
location, and under the same regulation.

The DNA to be inserted is flanked by sequences

that are homologous to those at the target
location.
1. Insertional Vectors
2. Replacement of DNA

Targeting Vectors Rely on Homologous

Recombination

Deliberate control of transgene expression

Inducible Endogenous Promoters

Promoter is induced by heavy metals such as lead, cadm

The heat shock promoter from Drosophila Hsp70 gene.

Successful examples of transgenic animals.

Stable expressions of human proteins have been
developed in many animals, including sheep, pigs, and
rats. For example, Human-alpha-1-antitrypsin, which has
been developed in sheep and is used in treating humans
with this deficiency.
Transgenic pigs with human-histo-compatibility have
been studied in the hopes that the organs will be
suitable for transplant with less chances of rejection.
Transgenic livestock have been used as bioreactors
since the 1990s. Many medicines, including insulin and
many immunizations are developed in transgenic
animals.
In March 2011, the bioactive recombinant Human
Lysozyme was expressed in the milk of cloned
transgenic cattle.

In 1999, scientists at the University of Guelph in Ontario,

Canada created the genetically engineered Enviropig.
In 2009, scientists in Japan announced that they had
successfully transferred a gene into a primate species
(marmosets) and produced a stable line of breeding
transgenic primates for the first time. Their first
research target for these marmosets was Parkinson's
disease.
In 2011, scientists in China released news that they have
introduced human genes into 300 dairy cows to produce
milk with the same properties as human breast milk.
Aside from milk production, the researchers claim these
transgenic cows to be identical to regular cows.
This field is growing rapidly and new pharming uses are