TRANSGENIC

ANIMALS
TERM PAPER – CONCEPTS IN
BIOTECH

SUBMITTED TO: MR.HARSH

SUBMITTED BY:-

Amandeep Sharma
B.Tech Biotech
Sec:- B
Roll No:- 19
Group :- G1.
 ACKNOWLEDGEMENT
Nothing concrete can be achieved without an optimal of
inspiration and perspiration.

And thanking people who have contributed to guide me is a

little like saying thank you at the academic awards. But still I
must dilute our feeling enough so as to fit them into framework
of words and acknowledge .I would like to take a moment to
thank those invaluable suggestion and support led to the
completion of this Term Paper. I acknowledge the contribution
of our teacher Mr. Harsh.
Last but not least I am very grateful to all those who helped me
in one or other way at every stage of my work.

AMANDEEP SHARMA
INTRODUCTION:-

TABLE OF CONTENTS
1-TRANSGENIC ANIMALS
METHOD OF PRODUCING TRANSGENIC MICE
2-THE PRONUCLEUS METHOD
TRANSFORM FERTILIZED EGGS
3-KNOCKOUT MICE
4-TISSUE SPECIFIC KNOCKOUT MICE
5-THE CRE/LoxP SYSTEM
6-TRANSGENIC SHEEPS AND GOATS
7-TRANSGENIC CHICKENS
8-TRANSGENIC PIGS

TRANSGENIC ANIMALS
A transgenic animal is one that carries a foreign gene that
has been deliberately inserted into its genome. The foreign
gene is constructed using recombinant DNA methodology. In
addition to a structural gene, the DNA usually includes
other sequences to enable it

• To be incorporated into the DNA of the host and

• To be expressed correctly by the cells of the host.

• Transgenic sheep and goats have been produced that

express foreign proteins in their milk.
• Transgenic chickens are now able to synthesize human
proteins in the "white" of the eggs.

These animals should eventually prove to be valuable

sources of proteins for human therapy.

Transgenic mice have provided the tools for exploring many

biological questions.

An example:
Normal mice cannot be infected with polio virus. They lack
the cell-surface molecule that, in humans, serves as the
receptor for the virus. So normal mice cannot serve as an
inexpensive, easily-manipulated model for studying the
disease. However, transgenic mice expressing the human
gene for the polio virus receptor

• can be infected by polio virus and even

• Develop paralysis and other pathological changes
characteristic of the disease in humans.

Two methods of producing transgenic mice are widely

used:
• Transforming embryonic stem cells (ES cells)
growing in tissue culture with the desired DNA.

• Injecting the desired gene into the pronucleus of a

fertilized mouse egg.

The Embryonic Stem Cell Method (Method "1")

Embryonic stem cells (ES cells) are harvested from the
inner cell mass (ICM) of mouse blastocysts. They can be
grown in culture and retain their full potential to produce all
the cells of the mature animal, including its gametes.
1. Make your DNA

Using recombinant DNA methods, build molecules of DNA

containing

• The structural gene you desire (e.g., the insulin gene)

• Vector DNA to enable the molecules to be inserted into
host DNA molecules
• Promoter and Enhancer Sequences to enable the
gene to be expressed by host cells
2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will

incorporate it.

3. Select for successfully transformed cells.

4. Inject these cells into the inner cell mass (ICM) of

mouse blastocysts.

5. Embryo transfer

• Prepare a pseudo pregnant mouse (by mating a

female mouse with a vasectomized male). The stimulus
of mating elicits the hormonal changes needed to make
her uterus receptive.
• Transfer the embryos into her uterus.
• Hope that they implant successfully and develop into
healthy pups (no more than one-third will).

6. Test her offspring

• Remove a small piece of tissue from the tail and

examine its DNA for the desired gene. No more than
10–20% will have it, and they will be heterozygous for
the gene.

7. Establish a transgenic strain

 • Mate two heterozygous mice and screen their offspring
for the 1:4 that will be homozygous for the transgene.

3. Implant the embryos in a pseudo pregnant foster

mother and proceed as in Method 1.

An Example
This image (courtesy of R. L.
Brinster and R. E. Hammer) shows
a transgenic mouse (right) with a
normal littermate (left). The giant
mouse developed from a fertilized
egg transformed with a
recombinant DNA molecule
containing:

• The structural gene for human growth hormone

• A strong mouse gene promoter
• The levels of growth hormone in the serum of some of
the transgenic mice were several hundred times higher
than in control mice.

Random vs. Targeted Gene

Insertion
The early vectors used for
gene insertion could, and did,
place the gene (from one to 200 copies of it) anywhere in
the genome. However, if you know some of the DNA
sequence flanking a particular gene, it is possible to design
vectors that replace that gene. The replacement gene can
be one that

• Restores function in a mutant animal or

• Knocks out the function of a particular locus.

In either case, targeted gene insertion requires

• the desired gene

• neor, a gene that encodes an enzyme that inactivates
the antibiotic neomycin and its relatives, like the drug
G418, which is lethal to mammalian cells;

tk, a gene that encodes thymidine kinase, an enzyme that

phosphorylates the nucleoside analog gancyclovir.

• DNA polymerase fails to discriminate against the

resulting nucleotide and inserts this nonfunctional
nucleotide into freshly-replicating DNA. So ganciclovir
kills cells that contain the tk gene.

Step 1

Treat culture of ES cells with preparation of vector DNA.

Results:
 • Most cells fail to take up the vector; these cells will be
killed if exposed to G418.
• In a few cells: the vector is inserted randomly in the
genome. In random insertion, the entire vector,
including the tk gene, is inserted into host DNA. These
cells are resistant to G418 but killed by gancyclovir.
• In still fewer cells: homologous recombination occurs.
Stretches of DNA sequence in the vector find the
homologous sequences in the host genome and the
region between these homologous sequences replaces
the equivalent region in the host DNA.

Step 2

Culture the mixture of cells in medium containing both G418

and ganciclovir.

• The cells (the majority) that failed to

take up the vector are killed by
G418.
• The cells in which the vector was
inserted randomly are killed by
gancyclovir (because they contain
the tk gene).
• This leaves a population of cells
transformed by homologous
recombination (enriched several
thousand fold).
Step 3

Inject these into the inner cell mass of mouse blastocysts.

Knockout Mice: What do they teach us?

If the replacement gene (A* in the diagram) is nonfunctional

(a "null" allele), mating of the heterozygous transgenic mice
will produce a strain of "knockout mice" homozygous for
the nonfunctional gene (both copies of the gene at that locus
have been "knocked out").

Knockout mice are valuable tools for discovering the

function(s) of genes for which mutant strains were not
previously available. Two generalizations have emerged
from examining knockout mice:

• Knockout mice are often surprisingly unaffected by

their deficiency. Many genes turn out not to be
indispensable. The mouse genome appears to have
sufficient redundancy to compensate for a single
missing pair of alleles.
• Most genes are pleiotropic. They are expressed in
different tissues in different ways and at different times
in development.
Tissue-Specific Knockout Mice

While “housekeeping genes” are expressed in all types of

cells at all stages of development, other genes are normally
expressed in only certain types of cells when turned on by
the appropriate signals (e.g. the arrival of a hormone).

To study such genes, one might expect that the methods

described above would work. However, it turns out that
genes that are only expressed in certain adult tissues may
nonetheless be vital during embryonic development. In such
cases, the animals do not survive long enough for their
knockout gene to be studied.

Fortunately, there are now techniques with which transgenic

mice can be made where a particular gene gets knocked out
in only one type of cell.

The Cre/loxP System

One of the bacteriophages that infects E. coli, called P1,

produces an enzyme — designated Cre — that cuts its DNA
into lengths suitable for packaging into fresh virus particles.
Cre cuts the viral DNA wherever it encounters a pair of
sequences designated loxP. All the DNA between the two
loxP sites is removed and the remaining DNA ligated
together again (so the enzyme is a recombinase).

Using "Method 1", mice can be made transgenic for

 • the gene encoding Cre attached to a promoter that will
be activated only when it is bound by the same
transcription factors that turn on the other genes
required for the unique function(s) of that type of cell;

a "target" gene, the one whose function is to be studied,

flanked by loxP sequences.

The result: a mouse with a particular gene knocked out in

only certain cells.

Knock-in Mice

The Cre/loxP system can also be used to

• Remove DNA sequences that block gene transcription.

The "target" gene can then be turned on in certain cells
or at certain times as the experimenter wishes.
• Replace one of the mouse's own genes with a new gene
that the investigator wishes to study.

Such transgenic mice are called "knock-in" mice.

Transgenic Sheep and Goats

Until recently, the transgenes introduced into sheep inserted

randomly in the genome and often worked poorly. However,
in July 2000, success at inserting a transgene into a specific
gene locus was reported. The gene was the human gene for
alpha1-antitrypsin, and two of the animals expressed
large quantities of the human protein in their milk.

This is how it was done.

Sheep fibroblasts growing in tissue culture were treated with

a vector that contained these segments of DNA:

1. 2 regions homologous to the sheep COL1A1 gene. This

gene encodes type 1 collagen. (Its absence in humans

causes the inherited disease osteogenesis imperfect.)

This locus was chosen because fibroblasts secrete large

amounts of collagen and thus one would expect the
gene to be easily accessible in the chromatin.

2. A neomycin-resistance gene to aid in isolating those

cells that successfully incorporated the vector.
3. The human gene encoding alpha1-antitrypsin.

Some people inherit two non- or poorly-functioning

genes for this protein. Its resulting low level or absence
produces the disease Alpha1-Antitrypsin Deficiency
(A1AD or Alpha1). The main symptoms are damage to
the lungs (and sometimes to the liver).

4. Promoter sites from the beta-lactoglobulin gene.

These promote hormone-driven gene expression in

milk-producing cells.
 5. Binding sites for ribosome for efficient translation of the

mRNAs.

Successfully-transformed cells were then

• Fused with enucleated sheep eggs and

• Implanted in the uterus of a ewe (female sheep).
• Several embryos survived until their birth, and two
young lambs have now lived over a year.
• When treated with hormones, these two lambs secreted
milk containing large amounts of alpha1-antitrypsin
(650 µg/ml; 50 times higher than previous results using
random insertion of the transgene).

On June 18, 2003, the company doing this work abandoned it

because of the great expense of building a facility for
purifying the protein from sheep's milk. Purification is
important because even when 99.9% pure, human patients
can develop antibodies against the tiny amounts of sheep
proteins that remain.

However, another company, GTC Biotherapeutics, has

persevered and in June of 2006 won preliminary approval to
market a human protein, antithrombin, in Europe. Their
protein — the first made in a transgenic animal to receive
regulatory approval for human therapy — was secreted in
the milk of transgenic goats.

Transgenic Chickens
Chickens

• Grow faster than sheep and goats and large numbers

can be grown in close quarters;
• Synthesize several grams of protein in the "white" of
their eggs.

Two methods have succeeded in producing chickens

carrying and expressing foreign genes.

• Infecting embryos with a viral vector carrying

o The human gene for a therapeutic protein
o Promoter sequences that will respond to the
signals for making proteins in egg white.
• Transforming rooster sperm with a human gene and the
appropriate promoters and checking for any transgenic
offspring.

Preliminary results from both methods indicate that it may

be possible for chickens to produce as much as 0.1 g of
human protein in each egg that they lay.

Not only should this cost less than producing therapeutic

proteins in culture vessels, but chickens will probably add
the correct sugars to glycosylated proteins — something that
E. coli cannot do.

Transgenic Pigs

Transgenic pigs have also been produced by fertilizing

normal eggs with sperm cells that have incorporated foreign
DNA. This procedure, called sperm-mediated gene transfer
(SMGT) may someday be able to produce transgenic pigs
that can serve as a source of transplanted organs for
humans.

Applications of transgenic animals:-

 Transgenic animals can be

produced following transfer of
cloned DNA into fertilized oocytes
and cells from very early stage
embryos
 Cultured embryonic stem (ES) cells
provide a powerful route to genetic
modification of the germline
  Transgenic animals have been used for
a variety of studies investigating
mammalian gene expression and
function

 The use of YAC transgenes and

inducible promoters has greatly
extended the applications of transgenic
animals.

REVIW OF LITERATURE:-

1.Genetically modified organism (GMO):-

As described by Holst-Jensen (2001) genetically modified

organism (GMO) is a living organism, e.g. bacteria, plant,
animal, whose genetic composition has been altered by
means of gene technology. The genetic modification usually
involves insertion of a piece of DNA and/or synthetic
combination of several smaller pieces of DNA, into the
genome of the organism to be modified. This process is
called transformation. These DNA pieces are usually taken
from other organisms such as bacteria or virus. A typical
insert (gene construct) in a GMO is composed of three
elements: 1) The promoter element functions as an on/off
switch for reading of the inserted gene(s); 2)The gene(s) that
has been inserted, which coding for a specific selected
feature; 3) The terminator element functions as a stop signal
for reading of the inserted gene(s). In addition, several other
elements can be present in a gene construct, and their
function is usually to control and stabilize the
function of the gene, demonstrate the presence of
the construct in the GMO or facilitate combination of
the various elements of the construct. The area
planted with genetically modified (GM) crops, including
soybeans and maize has increased worldwide in recent
years. Between 1996 and 2002, it rose from 1.6 × 106 to
more than 58 × 106 hectares (James, 2002) (Figure 2).
Recent report released by James (2003) mentioned that
worldwide plantings of biotech crops increased 12 percent in
2002. Between 5.5 million and 6 million farmers in 16
countries planted biotech seeds in 2002, up from 5 million
farmers in 13 countries in 2001. By the same way biotech
plantings will likely increase.
Among the other interestingly findings in this report:
• The area planted with biotech crops increased 35-fold
between 1996 and 2002.
• Four countries accounted for 99% of the global biotech
acreage [Argentina(23%), Canada (6%), China (4%) and the
United States (63%)]. The other 12countries accounted for
the remaining 1% [Australia, Bulgaria]
· The three new countries to begin planting biotech crops in
2002 were Colombia,Honduras and India. Columbia and
Honduras conducted “pre-commercial” biotech plantings in
anticipation of full commercial approval in 2003.
· Four crops accounted for 99% of the global biotech
plantings: Soybeans (63%), maize (19%), cotton (13%) and
canola (5%). Biotech squash and papaya each accounted for
less than 1%.
This rapid increase has provoked an explosion of concern
over the health and environmental impacts of these crops.
Despite claims of safety and warnings against popular panic,
public concern over GM crops has resulted in changes in
their marketing, labelling, planting, and trade.

2. Actually present and future of genetically

modified crops:-
Gaede (1997) and Flachowsky et al. (2002) classified edible
crops developed by modern biotechnology from the
nutritional point of view into two generations:
Worldwide production of GM soybeans and maize 1996-2002
(James, 2002)
- 1st Generation: Feed plants are characterized by
changed tolerance or resistance to insects, herbicides,
pesticides or other influencing factors with minor changes in
nutrient content (e.g. Bt-maize, Pat-maize, Roundup Ready
soybeans™, etc.).
- 2nd Generation: Feeds are characterized by
substantial changes in the content of valuable or undesirable
major ingredients (e.g. protein, amino acids, fat, fatty acids,
starch, sugars, lignin, etc.) or minor ingredients (e.g.
vitamins, minerals, enzymes, antinutritive
substances,etc.).The following overview (Matissek, 1998)
summarized the objects of molecular biological modifications
in plants with respect to food and feed production.The
objects of previous as well as prospective research work
allow to distinguish 4 groups:
A) Direct increase in yield by creating high productive
varieties of plants.
B) Enhancement of productivity by elimination or reduction
of disturbing biotic andabiotic influences and limitations, for
instance:
• introduction of herbicide resistance.
• control of pest and diseases.
• reduced losses due to abiotic stresses.
C) Improvement of post harvest management and storage
features.
D) Improvement of food and feed qualities by:
• direct improvement of nutritive value through modulation
of nutrient synthesis.
• elimination of unfavorable ingredients and increase
contents of health benefit substances.
3. Insect resistant Bt-maize:-
Shah et al. (1995) estimated the worldwide annual costs of
chemical control of maize plant insect (European corn borer
insecticides) at 3-5 billion US $. In addition, great losses in
maize plant yield are still caused by insect pests. To
overcome this economical as well as ecological impact of
chemical insecticides and to reduce the permanent danger
of residue accumulation of insecticides, genes encoding for
the insecticidal protein of Bacillus thuringensis (Bt) have
been inserted into maize and other crop species by
biotechnological methods to increase the resistant of the GM
plants to insect
pests (Ives, 1996). Bt (Bacillus thuringiensis) is a naturally
occurring soil bacterium that is found worldwide. A unique
feature of this bacterium is its production of crystal-like
proteins (Cry proteins) that selectively kill specific groups of
insect larvae (Bt-delta-endotoxins). Gasser and Fraley (1989)
reported that all types of Cry proteins revealed no toxicity to
beneficial insects, animals or human.

RESEARCH METHODOLOGY:-

Abstract:-- The notion of directly introducing new genes

or otherwise directly manipulating the genotype of an animal
is conceptually straightforward and appealing because of the
speed and precision with which phenotypic changes could be
made. Thus, it is of little wonder that the imagination of
many an animal scientist has been captivated by the success
others have achieved by introducing foreign genes into mice.
The private sector has embraced transgenic livestock
technology resulting in the formation of two new industries.
However, before transgenic farm animals become a common
component of the livestock production industry, a number of
formidable hurdles must be overcome.

FUTURE PROSPECTIVE :-

Male infertility has been considered a major contributory

factor to infertility. The causes of spermatogenetic
failure found in most cases of male infertility remain
largely idiopathic. Unfortunately, there is no effective
treatment to improve spermatogenesis for idiopathic
male infertility patients. In tracytoplasmic sperm
injection (ICSI) is the current treatment of choice for
severe male infertility and has brought the joy of
childbearing to couples for whom it was previously
impossible; however, several problems exist with this
treatment. In addition, if there are no spermatozoa in
the testis of these patients, they do not have paternity
potential even if ICSI is conducted. Ultimately,
fertilization is better in vivo than in vitro. Recently, on
the other hand, gene transfer to sperm and testis has
been developed to find more effective and simple
methods to obtain transgenic animals. This technique
has the potential to be the most useful approach for the
future treatment of male infertility. In this review, we
will give an overview of the recent advanced technique
of gene transfer to sperm and testis, and discuss the
future prospects of gene therapy for the treatment of
male infertility. In conclusion, although more
investigations on the mechanism of spermatogenesis
and male infertility and the establishment of techniques
for more efficient and safer gene transfer to the sperm
and testis will be needed, gene therapy will enable a
revolutionary advance for reproductive treatment and
 provide great benefit for patients with male infertility in
the future.

BIBLIOGRAPHY:-

WEB LINKS -
www.google.com

www.biospectrum.com

www.lifesciences.com

BOOKS -
Microbiology by Prescott.

Concepts in Biotech By B.D.Singh.

Basics of Animal Tissue Culture By Butler.