The document presents a detailed overview of transgenic animals, which are organisms that have been genetically modified to carry foreign genes for various purposes, including increased productivity in agriculture and advancements in medical research. It outlines methods for creating transgenic animals, historical milestones, advantages and disadvantages of specific species, and applications such as xenotransplantation and gene therapy. The document emphasizes the potential for transgenic technology to enhance traits in livestock and serve as models for human diseases, while also addressing challenges and ethical considerations.

1. BIOTECHNOLOGICAL
INTERVENTIONS IN FOOD SCIENCE
Presentation on
• METHODS FOR PRODUCING TRANSGENIC ANIMALS
SUBMITTED TO-
DR. Vandana Mam
SUBMITTED BY-
SOURABH
B.TECH. BIOTECHNOLOGY SRP
GF/2020/3377
3RD YEAR ( 6TH SEMESTER)

2. TRANSGENIC ANIMALS AND
METHODS FOR PRODUCING
TRANSGENIC ANIMALS

3. TRANSGENIC ANIMALS-
• Organisms containing integrated sequences of cloned DNA transferred using techniques of
genetic engineering are called transgenic animals. Or an Organism that is engineered to carry
a foreign gene or a transgene of choice as part of its own genetic material.
• In order to change the animal's DNA, a foreign gene is inserted into its genome. This
technique is applied to enhance the genetic traits of the target animals.
• Transgenic animals are altered so that their DNA produces proteins that normally they would
not produce or as by their non-genetically modified counterparts. Ex.- glow fish as pets,
sheep with more wool, a cow producing more milk with lower cholesterol.
• Transgenic animals with changes in the germ line are heritable from generation to generation
within the herd, and this heritability has the potential to facilitate long-term productivity
gains.

4. • Process of introducing foreign or exogenous DNA into animals genome is called
transgenesis or transgenesis is the process of introducing an exogenous gene called a
transgene into a living organism so that the organism will exhibit a new property and
transmit that property to its offspring.
• Transgenesis can be facilitated by liposomes, enzymes, plasmid vectors, viral vectors,
pronuclear injection, protoplast fusion, ballistic DNA injections, etc. Transgenic technology
has led to the development of fishes livestock and other animals with altered genetic profiles
which are useful to mankind. Examples for genetically modified animals: Mice, Goat, Sheep,
Chicken, Cow, Horse, Dogs, Fish, etc.

5. HISTORY-
• The first genetically modified Organism was created by Stanley Cohen and Herbert Boyer
a bacteria in 1973 followed by the first transgenic animal in 1974 that were mice created
in the 1970s by Rudolph Jaenisch a professor of biology at Massachusetts Institute of
technology.
• In the year 1982 worlds first expressing transgenic animal super mouse was produced by
inserting a human growth hormone gene into the mouse genome.

6. ANIMALS-
1.TRANSGENIC COW
Transgenic cows carrying extra copies of two types of casein genes produce 13%
more milk protein. Currently, the milk from these animals is under FDA review.
2. ENVIRO PIG:
They are used in organ transplant harvesting and study of human membrane co-
factor protein. Breeding problems and Mutation will occur.
3.TRANSGENIC FISH
Tilapia, Salmon/trout, Catfish
These can grow up to 6 times faster than wildtype fish and most have extra copies
of growth hormone (GH) gene

7. 4. Transgenic sheep:
Tracy is the first transgenic animal to produce a recombinant protein in her milk. uses of transgenic
sheep: It is used as a model for studying Human blood clotting factor viii, Transplantation, Haematology
Biological product manufacturing, Recombinant DNA, Drug production in milk. Disadvantages-
Difficult procedure, Failed in vitro fertilization, Expensive
5. Transgenic Monkey:
Its so similar to human hence it used in clinical trial used for studying: HIV, Huntington's disease
Disadvantages- Expensive, Difficulty in Breeding problem
6. Transgenic mice:
ADVANTAGES- Gene mutation, Alzheimer's disease, Hypertension, Atherosclerosis, Cardiac
hypertrophy, Human leukocyte antigen Human gastric carcinoma, Making poliovirus vaccine, HIV
studies, Different type of cancer.
DISADVANTAGES: Expensive, Gene can only be added,not deleted, Embryos are not easily accessible
for manipulation

8. Production of GMOs is a multistage process which can be
summarized as follows:
1. Identification of the gene interest;
2. Isolation of the gene of interest;
3. Amplifying the gene to produce many copies;
4. Associating the gene with an appropriate promoter and poly A sequence and insertion into
plasmids.
5. Multiplying the plasmid in bacteria and recovering the cloned construct for injection;
6. Transference of the construct into the recipient tissue, usually fertilized eggs;
7. Integration of gene into recipient genome;
8. Expression of gene in recipient genome; and
9. Inheritance of genes through further generations.

9. GENE TRANSFER (GT):-
The introduction of new DNA into an existing organism’s cell, usually by vectors such as
plasmids and modified viruses. The transfer of genes between species is called GENE
TRANSFER. The new organism created is called transgenic. The insertion of unrelated
therapeutic genetic information in the form of DNA into target cells.
The directed desirable gene transfer from one organism to another and the subsequent
stable integration and expression of foreign gene into the genome is referred to as genetic
transformation. Transient transformation occurs when DNA is not integrated into host
genome.
Two Types of gene transfer:-
1. HORIZONTAL GT- Horizontal gene transfer can be described as the transfer of
genetic information between two independent organisms.
2. VERTICAL GT- Vertical gene transfer is the transfer of genetic information from
parent to progeny. Germline, Live birth, Patrilineal, Aposymbiotic.

10. These are divided into two main groups
• Indirect method: In this case vector is needed for the insertion of the foreign DNA into the
host genome.
• Direct method: This method is vector independent. The DNA is directly inserted into the host
genome.
Gene transfer
Direct/Artificial
physical
microinjection
Biolistics- gene
gun
Pollen
transformation
Micro laser
chemical
Poly-ethylene
glycol
Silicon carbide
method
Calcium
phosphate
Lipsomes and
cationic lipids
(lipofection)
Electrical
Electroporation
indirect
Biological
Agrobacterium
mediated
Virus mediated
Conjugation Transformation

11. METHODS/TECHNIQUES FOR PRODUCING TRANSGENIC ANIMALS-
• The main principle in the production of
transgenic animals is the introduction of a
foreign gene or genes into an animal (the
inserted genes are called transgenes).
• The foreign genes must be transmitted
through the germ line, so that every cell,
including germ cells, of the animal contains
the same modified genetic material.
• The first transgenic animals produced in
1980, were mice.
• There are various methods for producing
transgenic animals which are summarized in
the following figure.

12. Retrovirus Mediated Transfer-
• Transgenic mice produced by retroviral transduction of male germ line stem cells.
• Male germ line stem cells have ability to self-renew and genetic modification of these cells
would help to study the biology of their complex self-renewal and differentiation processes
and to generate wide range of transgenic animal species.
• A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA.
• The method was successfully used in 1974 when a simian virus was inserted into mice
embryos, resulting in mice carrying this DNA.
• In this method gene transfer is mediated by a carrier or vector. Its transfer own genetic
material into the cell, taking advantage of their ability to infect host cells. Offspring
consequential from this method are chimeric, i.e., not all cells carry the retrovirus. The killed
virus is replication defective.
• The virus gene is replaced with trans-gene is inserted to the host cell by transfection. This can
be used to transfect a wide range of cells such as embryonic cells. The outcome is chimera,
an organism consisting of tissues or parts of diverse genetic constitutions.

13. • Retro viral vectors that infects the cells of an early stage embryo prior to implantation into a receptive
female.
Technique:
• Immediately following infection, the retrovirus produces a DNA copy of its RNA genome using
transcriptase. Completions of this process require that the host cell undergoes the S phase of the cell
cycle. Therefore, retroviruses effectively transducer only mitotically active cell.
• The DNA copy of the viral genome, provirus, integrates randomly into the host cell genome, usually
without deletions or rearrangements because assimilation is not by way of homologous
recombination. Depending on the technique used, the Generation may result in chimeras. When the
transgene has integrated into the germ cells, the so-called germline chimeras are then inbred for 10 to
20 generations until homozygous transgenic animals are obtained and the transgene is present in
every cell.
• Current research has shown that lentiviruses can overcome previous limitations of viral-mediated
gene transfer, which included the silencing of the transgenic locus and low expression levels.
Injection of lentiviruses into the perivitelline space of porcine zygotes resulted in a very high
proportion of piglets that carried and expressed the transgene. Stable transgenic lines have been
established by this method.

14. Retroviral vectors into germ line (8-
cell embryo infected) transgenic
animals-

15. Microinjection-
1. Pronuclear -
• The DNA microinjection or pronuclear microinjection, a very fine glass pipette is used to
manually inject DNA from one organism into the eggs of another.
• Better time for injection is early after fertilization when the ova have two pronuclei. They
fused to form a single nucleus, the injected DNA may or may not be taken up.
• Through the DNA microinjection, the ovum is transferred into the oviduct of the recipient
female or foster mother that has been induced by mating with a vasectomized male. The
University of California (Irvine) Transgenic Mouse Facility reports an estimated success rate
of 10% to 15% based on experiments with mice testing positive for the transgene.
• If the DNA is assimilated into the genome, it is done so randomly.
• Because of this, there is always a chance the gene insert will not be expressed by the GMO,
or may even interfere with the expression of another gene on the chromosome

16. 2. Embryonic Stem cell-mediated gene transfer-
• In 1981, the term embryonic stem cells (ES cells) were used to denote a cell line isolated
directly from mouse embryos while the term embryonal carcinoma cells (EC) were derived
from teratocarcinomas.
• 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.
Technique-
• 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.
• Transform ES cells in culture to expose cultured cells to the DNA so that some will
incorporate it. Select successfully transformed cells. Inject these cells into the inner cell mass
(ICM) of mouse blastocysts.

17. • Embryo transfer:
- Prepare a pseudopregnant mouse. 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).
• 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.
• Establish a transgenic strain:
- Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous
for the transgene.

18. Embryonic stem cell method for
producing transgenic mice-
Pronuclear microinjection and ES cells
mediated transfer-

19. Somatic Cell Nuclear Transfer (SCNT)-
• In this method, the transgenic goats were produced by nuclear transfer of fetal somatic cells.
Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day
transgenic female fetus produced by artificial insemination of a nontransgenic adult female with
semen from a transgenic male. Live offspring were produced with two nuclear transfer
procedures.
• Oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic
cells, and simultaneously activated.
• In the second procedure, activated in vivo oocytes were enucleated at the telophase II stage,
electrofused with donor somatic cells, and simultaneously activated a second time to induce
genome reactivation.
There was generation of three healthy identical female offspring. Genotypic analyses confirmed that
all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the
transgenic cloned animals showed high-level production of human antithrombin III. The nuclear
transfer application may be more useful and beneficial for agricultural is the ability to efficiently
produce a large number of identical offspring derived from a particular mating. Therefore, nuclear
transfer using a embryonic cell lines derived from that mating maybe more attractive.

20. Dolly sheep –
• Dolly was a female domestic sheep, and the first mammal cloned from an adult somatic cell,
using the process of nuclear transfer. Born 5 July 1996. She was cloned by Sir lan Wilmut,
Keith Campbell and colleagues at the Roslin Institute, part of the University of Edinburgh,
Scotland.
• In this process, the udder cells from a Finn Dorset white sheep of 6 years old were injected
into an unfertilized egg from a Scottish Blackface ewe, whose nucleus was removed.
• With the help of electrical pulses, the cell was made to fuse.
• After fusion of the cell nucleus with the egg, the resulting embryo was cultured for the next
6-7 days.
• The embryo was then implanted into another Scottish Blackface Ewe, which was responsible
for the birth of Transgenic Sheep, Dolly.

22. APPLICATIONS-
(A.) Agricultural Applications
(a). Breeding- Farmers have always used discriminatory breeding to produce animals that exhibit
desired traits (e.g. increased milk production, high growth rate). Traditional breeding is a time-
consuming, difficult chore. When expertise using molecular biology was developed, it became
possible to develop traits in animals in a shorter time and with more precision. In sum, it offers the
farmer an easy way to increase yields.
(b). Quality- Transgenic cows stay alive that produce more milk or milk with less lactose or
cholesterol, pigs and cattle that have more meat on them, and sheep that grow more wool. In the
past, farmers used growth hormones to stimulate the development of animals but this technique
was problematic, especially since the residue of the hormones remained in the animal product.
(c). Disease resistance- Scientists are attempting to produce disease-resistant animals, such as
influenza-resistant pigs, but an inadequate number of genes is currently known to be responsible
for resistance to diseases in farm animals.

23. (B). Medical Applications
(a). Xenotransplantation- Patients die every year to be deficient of replacement heart, liver, or
kidney. For example, about 5000 organs are needed each year in the United Kingdom alone.
Transgenic pigs may offer the transplant organs needed to improve the loss. Presently,
xenotransplantation is vulnerable by a pig protein that can cause donor rejection but research is
underway to remove the pig protein and replace it with a human protein. One of the earliest
genetic modifications of larger animals was the development of genetically modified pigs
transport a human gene that could prevent the acute rejection of organs transplanted between
pigs and humans. The transplantation of tissues from one species to a different species is known
as xenotransplantation. Whenever pig tissue is transplanted into another species, antibodies in
the receiver attack the transplanted organ, and the resulting inflammatory response leads to graft
rejection. By introducing a modification to some of the proteins on cells that cause the body to
raise an immune response, called balance control proteins, rejection of the transplant can be
disallowed.

24. (b). Nutritional supplement and pharmaceutical- Some Products such as insulin, growth
hormone, and blood anti-clotting factors may soon be or have already been obtained from the
milk of transgenic cows, sheep, or goats. Research is ongoing to manufacture milk through
transgenesis for treatment of unbearable diseases such as phenylketonuria (PKU), hereditary
emphysema, and cystic fibrosis. In 1997, foremost transgenic cow, Rosie, produced human
protein-enriched milk at 2.4 grams per liter. This transgenic milk is an additional nutritionally
balanced product than natural bovine milk and could be given to babies or the elderly with
unique nutritional or digestive needs. Rosie’s milk contains the human gene alpha-lactalbumin.
(c). Human gene therapy- Human gene therapy involves adding together a normal copy of a
gene (transgene) to the genome of a person carrying defective copies of the gene. The
prospective for treatments for the 5,000 named genetic diseases is huge and transgenic animals
could play a role. For example, the A.I.Virtanen Institute in Finland produced a calf with a gene
that makes the material that promotes the growth of red cells in humans.

25. (C). Transgenic Animals as Disease Models for the Development of New Treatments
An animal model is a, living, non-human animal used for the study and investigation of human
disease, for the purpose of better considering the disease without the added risk of causing harm
to a human being during the whole drug discovery and development process. Transgenic animal
models are created by the insertion of a particular human DNA into fertilized oocytes which are
then allowed to develop to term by implantation into the different models of transgenic animals
for various diseases oviducts of pseudo-pregnant females.
(a). Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome (HIV/AIDS)-
S Transgenic 26 human immunodeficiency virus associated nephropathy (Tg26 HIVAN) Mouse
Model was the first transgenic model developed in 1991 for the study of HIV. These transgenic
animals can express HIV-1 proteins; develop to symptoms and immune deficiencies similar to
the manifestations of AIDS in humans. Other models are AIDS Mouse and Smart Mouse.

26. (b). Alzheimer’s Disease- There wasno animal models existed for the disease before transgenic
technology was working. Immunization of Amyloid precursor protein (A42) in Pigs in
transgenic mice showed that vaccination against Alzheimer’s disease could have potential as a
therapeutic approach. E.g. Alzheimer’s Mouse.
(c). Cardiovascular Disease- Various transgenic animal several models for gain and or loss of
function of angiotensin, endothelim, natriuretic peptides, catechoalmines, calcium Binding-
signaling, sodium channel transporters and nitric oxide synthesis involved in cardiovascular
parameter are used to study cardiovascular diseases.
(d). Diabetes Mellitus- Transgenic model are developed for studying the genes and their role in
peripheral insulin accomplishment. Models of insulin secretion such as glucokinase, tislet
amyloid polypeptide and hepatic glucose production in type II diabetes are developed. 18A
model that expressed Insulin Dependent Diabetes Mellitus by inserting a viral gene in the
animal egg stage is also developed.

27. (D) Industrial Applications
Two scientists discovered (Dr. Jeffrey Turner & Randy Lewis) a sliced spider gene into the cells
of lactating goats in 2001 at Nexia Biotechnologies in Canada. The goats begin to produce silk
along with their milk and secrete tiny silk strands from their body by the bucketful. By
extracting polymer strands from the milk and weaving them into the strand, the scientists can
generate a light, tough, flexible material that could be used in such applications as military
uniforms, medical tiny caliber suture called (microsuture), and tennis racket strings. The
toxicity-sensitive transgenic animals have been produced for chemical safety testing.
Microorganisms have been engineered to produce spacious variety of proteins, which in turn
can produce enzymes that can speed up industrial chemical reactions.

28. THANK YOU