Transgenic pigs are genetically engineered to have desired traits. There are several methods used to create transgenic pigs, including microinjection of DNA into pig zygotes, retrovirus-mediated gene transfer, and somatic cell nuclear transfer. Transgenic pigs are studied as models for human diseases and could potentially be a source of organs for xenotransplantation. Key applications include using transgenic pigs to study cardiovascular diseases, wound healing, and as potential donors for heart transplants.

1. Transgenic animals WITH DESIRED TRAITS
D.INDRAJA

2. Transgenesis
• Transgenesis refers to the phenomenon of introduction of
exogenous DNA In to the genome to create and maintain
a stable heritable character
• The foreign DNA that is introduced is called trans gene
• And the animal whose genome is altered by adding one or
more trans genes is said to be transgenic
• The trans genes behave as other genes and these are
passed on to the off springs. Thus transgenic animals are
genetically engineered or genetically modified organisms
(GMOs) with a new heritable character.

3. WHY TRANSGENIC ANIMALS ARE
PRODUCED?
• To study promoter function ,reporter gene expression and functions of transferred
genes
• To produce new proteins from cultured cell lines ,by the insertion and expression of
genes coding for specific proteins
• To create transgenic animals with higher yield of milk or meat
• To obtain better quality milk ,meat ,wool , etc
• To produce transgenic animals with the capacity to synthesize new proteins .such
animals are used in gene Pharming (Pharming means to producing Pharmaceuticals
• To use animals as models for studies in gene therapy
Gene pharming is a technology that scientists use to alter an animal's own DNA, or to
splice in new DNA, called a transgene, from another species. In pharming, these
genetically modified (transgenic) animals are used mostly to make human proteins that
have medicinal value.

4. • Historical background
• Prior to the development of molecular genetics, the only way of studying the regulation
and function of mammalian genes was through the observation of inherited
characteristics or spontaneous mutations.
• Long before Mendel and any molecular genetic knowledge, selective breeding was a
common practice among farmers for the enhancement of chosen traits, e.g., increased
milk production.
Disadvantages
•Time consuming and costly
•Larger animals having long gestation
period it may take several decades to
create desired character by conventional
breeding

5. • During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two
different embryos of different strains were combined together at an early stage of
development (eight cells) to form a single embryo that subsequently developed into a
chimeric adult, exhibiting characteristics of each strain.
• mutual contributions of developmental biology and genetic engineering permitted rapid
development of the techniques for the creation of transgenic animals. DNA microinjection,
the first technique to prove successful in mammals, was first applied to mice (Gordon and
Ruddle, 1981) and then to various other species such as rats, rabbits, sheep, pigs, birds, and
fish.
• Two other main techniques were then developed: those of retrovirus-mediated transgenesis
(Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).

8. Mouse is regarded as researcher-friendly by
biotechnologists
•Mouse is the animal of choice for transgenic experiments due to
•Being a small animal-easily handled
•It produces more eggs(normal mouse 5-10 eggs where as super ovulated mouse produces
40 eggs)
• Maintained and adapt well to new surroundings
•Have short lifespan of 2-3 years
•Relatively inexpensive
•Mild-tempered and docile
•Medical trials are uniform
•Mimic human body

9. Transgenic mice
• The first animal used for transgenesis was mouse. The super mouse was created by inserting a
rat gene for human growth hormone in to the mouse genome .the offspring was much larger
than the parents
• There are three methods for introducing a foreign gene in to mice and infact the same
methods are applicable to other animals as well
• Retrovirus-Mediated Gene Transfer
• Micro injection method
• Embryonic stem cell method

10. construction of a transgene
Transgene made of three parts-
• Promoter
• Gene to be expressed
• Termination sequence
Retrovirus-Mediated Gene Transfer
• A retrovirus is an animal virus that contain RNA that encodes for DNA.
• Retroviruses transfer genetic material into the host cell to produce Chimeras- animal
with mixed DNA
• To increase the probability of expression, gene transfer is mediated by means of a carrier
or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to
transfer genetic material into the cell, taking advantage of their ability to infect host cells
in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the
retrovirus.
• Transmission of the transgene is possible only if the retrovirus integrates into some of
the germ cells.

11. Disadvantages
• The transfer of small pieces of DNA (8kb) can be effectively carried out by retro viruses
• This method however is unsuitable for transfer of larger genes. Further even for small genes
there is loss of some regulatory sequences.
• Above all the biggest draw back is the risk of retroviral contamination in the products
obtained from transgenic animals (particularly in foods for human consumption)
• Because of this limitations the retro viral vector method is not in regular use for transgenesis

12. Micro injection method
The young virgin female mice(4-5 weeks age) are subjected to super ovulation
The super ovulated mice are mated with males and sacrificed on the following day
and the fertilized eggs are removed from the fallopian tube
By micromanipulation using a micro injection needle and a holding pipette the DNA is
injected in to the male pronucleus
Of the fertilized egg ( adequate care must be taken to ensure that while the elastic
nuclear membrane is punctured the needle does not touch the nucleoli)
The eggs with the transgenes are kept overnight in an incubator to develop to a 2 cell stage
These eggs are then implanted microsurgically in to a foster mother and it can
deliver pups after 3 weeks of implantation

13. Key terms
• Super ovulation- it is achieved by administration of follicle stimulating hormone
(pregnant mare’s serum) followed by (2 days later) human chronic gonadotropin
• Identification of male pronucleus- a dissection microscope is used to identify male
pronucleus which is larger in size
• Foster mother- pseudo mouse pregnant female mouse which has been mated the
previous night with vasectamized (infertile) male. The stimulus of mating elicits the
hormonal changes needed to make her uterus receptive.
•The presence of transgene in the pups can be identified by polymerase chain
reaction or southern blot hybridization

15. Disadvantages
• The microinjection method involves several steps and none of them is 100% efficient for any
animal to develop in to transgenic animal
• The foreign DNA randomly integrates in to the host genome
• Sometimes even many pieces of DNA get incorporated at a single site .further transgenes may
not be expressed at all or some times under expressed or over expressed –this will disturb the
normal physiology of the animal
• Time consuming
• Costly and labour intensive
Despite all these limitations this technique is routinely used for producing transgenic animals

16. Embryonic stem cell method
Prepare a recombinant DNA that should insert which contains a promoter enhancer,
silencer and desired gene
Expose the cultured embryonic stem cells to the DNA so that some will incorporate it.
Select for sucessfully transformed cells
Tranfer the embryo in to the uterus of a pseudo pregnant mouse
Inject these cells in to inner cell mass of a blastocyst
Test her off spring for the recombinant gene after delivering the pups

17. Key terms
embryonic stem cells -Cells from the inner cellmass of the blastocyst stage
of a developing mouse embryo can proliferate in cell culture
Pluripotent stem cells- Stem cells are undifferentiated cells that have the
potential to differentiate into any type of cell (somatic and germ cells) and
therefore to give rise to a complete organism.

18. Desired DNA
Select for cells expressing desired gene
Inject transformed ES cells tin to inner cell
mass
Implant in to uterus of a foster mother
and test the offsprings
Embryoni
c stem
cell
method

19. Selection of transgene containing cells
Pathways for synthesizing nucleotides
Thymidine kinase- used as a marker gene

20. • Dolly the first ever mammal clone was developed by wilmut and campbell in 1997
• It is a sheep (female lamb) with a mother and no father
• The technique primarily involves nuclear transfer and the phenomenon of totipotency
Key terms
• The cells of an adult lack totipotency it was induced in to the adult cells for developing
dolly
Success
• As reported by wilmut and campbell they fused 277 ovum cells, achieved 13
pregnancies, and of these only one pregnancy resulted in live birth of the offspring only
TRANSGENIC SHEEP
Totipotency/pluripotency  it is the basic character of embryonic cells . As
the embryo develops te cells specialize to finally give the whole organism

21. NUCLEAR TRANSFER IN
SHEEP
Making Dolly
– Treat the ewes with gonadotropin-releasing hormone (GnRH) to cause them to
produce oocytes ready to be fertilized. Like all mammals, these are arrested at
metaphase of the second meiotic division (meiosis II).
– Plunge a micropipette into the egg over the polar body and suck out not only the
polar body but the haploid pronucleus within the egg.
– Cells from the mammary gland of an adult Finn Dorset ewe (they have white faces) are
grown in tissue culture.
– Five days before use, the nutrient level in the culture is reduced so that the cells stop
dividing and enter G0 of the cell cycle.
– Donor cells and enucleated recipient cells are placed together in culture.
– The cultures are exposed to pulses of electricity to
• cause their respective plasma membranes to fuse;
• stimulate the resulting cell to begin mitosis (by mimicking the stimulus of
fertilization).
Enucleate the eggs produced by Scottish Blackface ewes (female sheep).
Fuse each enucleated egg with a diploid cell growing in culture.

22. • Culture the cells until they have grown into a morula (solid mass of cells) or even into
a blastocyst (6 days).
• Transfer several of these into the uterus of each (of 13, in this case) Scottish Blackface
ewes (previously treated with GnRH to prepare them for implantation.
• Wait (with your fingers crossed).
• The result: one ewe gave birth (148 days later) to Dolly

23. TRANSGENIC SHEEP
APPLICATIONS
• Some of the pharmaceutical products produced in the milk of transgenic
sheep are
• Clotting factors
• Soluble cd4 protein
• Lactoferin
• Urokinase
• CFTR
• Interleukin-2
• And high rate of milk,wool and meat yielding is seen
• 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.
• Successfully-transformed cells were thenfused with enucleated sheep eggs
and
• implanted in the uterus of a ewe (female sheep).
• Several embryos survived until their birth, and two young lambs lived over a
year.

25. TRANSGENIC COW
Method  (nuclear transfer) by using fetal cells
• Fetal cells such as fibroblasts are totipotent
• Fetal cell cloning was successfully carried out by some workers to produce transgenic sheep,
transgenic bull calf and other animals
Fibroblasts were collected from a fifty five day old bovine fetus and are cultivated in
nutritious medium
Desired foreign gene is introduced in to fibroblasts
Nucleus (with genetically altered DNA ) is taken out from the fibroblasts
Nucleus taken from fibroblst is fused with the enucleated ovum and it develop in to
embryo
Embryos are implanted in a surrogate (foster) mother cow to give birth to transgenic
calves

27. Transgenic Cow Applications
• Carry extra copies of two types of Casein genes
• 13% more milk protein
• Milk -more nutritious
• Currently the milk from these animals is under FDA review
Containing interferron protein which provide resistance against viral infections

28. Transgenic goats are developed with proteins containing proteins such as
•Cystic fibrosis transmembrane regulator (CFTR) for treatment of cystic fibrosis
•Tissue plasminogen activator  for treatment of myocardial infraction(dissolves blood clots)
•Anti thrombin III  for regulating blood clotting
TRANSGENIC GOAT

29. Transgenic pig
• Pigs, especially miniature pigs, have similar physiology to humans thus can
serve as an important biomedical model for human diseases
• The use of swine in biomedical research has gained much importance as
they have always been considered excellent models for the studies related to
various cardiovascular diseases, cutaneous pharmacology, diabetes, cancer
biology, lipoprotein metabolism, path biology of intestinal transport, injury
and repair, repair and healing of wounds, etc.
• Also been considered for being potential source of different organs for the
xenotransplantation as can be seen in the heart transplantation studies
• In 1985 First transgenic pig is created by by Microinjection of DNA into one
pronucleus of a zygote
Step 1 construction of a transgene
Transgene made of three parts-
• Promoter
• Gene to be expressed
• Termination sequence

30. • STEP 2:INTRODUCTION OF FOREIGN GENE
• There are a number of methods to carry out the genetic modification of the animals
Microinjection
• Injection of DNA construct directly into the pronuclei of zygotes Pronuclear injection is a
technique used to create transgenic organisms by injecting genetic material into the
nucleus of a fertilized oocyte.
Retrovirus-Mediated Gene Transfer
• To increase the probability of expression, gene transfer is mediated by means of a carrier
or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to
transfer genetic material into the cell, taking advantage of their ability to infect host cells
in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the
retrovirus.
• Transmission of the Transgene is possible only if the retrovirus integrates into some of
the germ cells.

31. Sperm-mediated gene
transfer
• A method highly efficient for the
transgenic pig creation, whereby the in-
vitro fertilization or insemination of the
pigs was carried out with sperm
previously mixed with DNA construct of
interest .
• The Genetic material is introduced into
sperm, which are used to fertilize eggs.
The embryos are carried to term. The
offspring may be transgenic.

32. somatic cell nuclear transfer (SCNT)
• In this technique in which the nucleus of a somatic (body) cell is transferred to the
cytoplasm of an enucleated egg (an egg that has had its own nucleus removed). Once
inside the egg, the somatic nucleus is reprogrammed by egg cytoplasmic factors to
become a zygote (fertilized egg) nucleus. The egg is allowed to develop to the blastocyst
stage, at which point a culture of embryonic stem cells (ESCs) can be created from the
inner cell mass of the blastocyst
Embryonic stem cell method
• Embryonic stem cells come from a five to six-day-old embryo. They have the ability to
form virtually any type of cell. Embryonic stem cells (ES cells) are harvested from the
inner cell mass of blastocysts. They can be grown in culture and retain their full
potential to produce all the cells of the mature animal, including its gametes. However,
this method has been successfully applied only in mice and for other species is yet to
be developed

33. Xenotransplantation
• Transplantation of living cells, tissues, and organs from one species to another is known
as xenotransplantation.
• Xenograft - is an organ transplanted from one species to another Human
xenotransplantation offers a potential treatment for end-stage organ failure, a significant
health problem in parts of the industrialized world.
• Xenotransplants could save thousands of patients waiting for donated organs.
Pig as an animal organ donor
• Easy to breed
• Pathogen free pig breeds are available
• Pig organs are similar to that of size of humans
• Risk of infection is lower in non human primates

34. Factors affecting Xenotransplantation are :
• Longevity
• Size
• Environment
• Hormone and protein differences
The Hyperacute rejection (HAR) of porcine xenografts is one of the
major constraints .Humans posses natural anti–pig antibodies that
are specific for alpha(1,3)-galactosyl epitopes on pig cells.
• Gal-alpha(1,3)-Gal is the proteins on the surface of pig cells but
not human ones.
• Attempts have been made to reduce the amount of this sugar
molecule by expressing antibodies against it, inhibiting the enzyme
that makes it (an enzyme called alpha-1,3-galactosyltransferase
that is only present in pigs) or using additional enzymes to modify
it.
• Most recently, two research groups have succeeded in completely
knocking out the alpha-1,3-galactosyltransferase gene, producing
pigs that cannot make this sugar at all.

35. Cloned transgenic pigs rich in omega-
3 fatty acids
• Polyunsaturated fatty acids (PUFAs) have 18 or more carbon atoms and two or more double
bonds.
• They can be classified into two groups, omega-6 (n-6) and omega-3 (n-3).
• Many studies in the last 20 years have shown the high n-6/n-3 PUFA ratio may contribute to
the high prevalence of many modern diseases (e.g., heart disease, autoimmune disorders, and
depression)
• Furthermore, the n-3 and n-6 PUFAs are not interconvertible in mammalian cells because
mammals also lack the enzyme, omega-3 fatty acid desaturase, to convert n-6 PUFA to n-3
PUFA
• An n-3 fatty acid desaturase gene, fat-1, was cloned from a roundworm .Expression of the fat-
1 gene in plants and mammalian cells showed FAT-1 protein converted n-6 PUFA to n-3
PUFA efficiently.
• A humanized fat-1 gene with the optimized codons for mammals was used to increase the
hfat-1 gene expression. The hfat-1 transgenic pig is also a good large animal model. It can be
used to study the effect and the mechanism of n-3 PUFAs in prevention and treatment of
coronary artery disease, hypertension, diabetes, arthritis, other inflammatory or autoimmune
disorders, and cancer

36. ENVIROPIGS
• Enviropigs have genetically modified salivary glands, which
help them digest phosphorus in feedstuffs and reduce
phosphorus pollution in the environment
• Phosphorus is crucial for healthy growth in pigs. Unfortunately,
50 to 70 percent of the phosphorus in grain is in the form of
phytic acid, a compound indigestible by pigs.
• Because of this, many farmers have to supplement pig diets with
an enzyme called phytase.
• Phytase breaks down phytic acid and helps pigs digest more of
the nutrient.
• The transgenic pig synthesizes phytase in its salivary glands,
eliminating the need for additional supplements or enzymes in
the feed.
• By digesting more phosphorus, the Enviropig also produces less
phosphorus in its waste.

37. • The different applications of genetically modified pigs in medical field can be summarized as
follows:
• The production of human haemoglobin in the blood of transgenic pigs for isolation and
treatment of trauma patients is one of the interesting applications being studied.
• The production of Protein C, in- activator of certain human coagulation factors in the milk of
pigs has been studied. It has been found that the mammary epithelial cells of the pigs are
capable of making the coagulation factors VIII and IX biologically active due to post-
translational modifications.
• The transgenic pigs can be used as better models for different diseases such as Retinitis
pigmentosa, cardiovascular diseases: Fat-1, Diabetes, Alzheimer’s disease, cystic fibrosis,
Huntington’s disease by the introduction of different mutations in the genes involved in the
pathophysiology of the diseases.
• The transgenic pigs can be used for cell tracking with the introduction of genes expressing
different fluorescent proteins into the pigs. The stem cells expressing fluorescent proteins
isolated from these transgenic pigs can be used as molecular markers for the tracking of
various biological mechanisms.
• The production of human hepatocytes in transgenic pigs to help in the transplantation of the
regenerated human hepatocytes to patients of liver failure from the transgenic pigs shows
great promise.
• Transgenic pigs also have application in agriculture in the production and growth of pigs
whose meat are safe environmentally, lean and healthier for human consumption by the
introduction of different genes expressing growth hormones and to reduce pollution by
alteration in the composition of the carcass

38. TRANSGENIC FISH
• A transgenic fish is one that contains genes from another species. A transgenic fish is an
improved variety of fish provided with one or more desirable foreign gene for the purpose of
enhancing fish quality, growth, resistance and productivity.
• The transgenic fish are being promoted as the first marketable transgenic animals for human
consumption
A FEW FACTS TO KNOW ABOUT TRANSGENIC FISH:
• Typically, genes of one or more donor-species are isolated, and spliced into artificially
constructed infectious agents, which act as vectors to carry the genes into the cells of
recipient species. Once inside a cell, the vector carrying the genes will insert into the cell’s
genome.
• transgenes have been introduced by microinjection or electroporation of DNA into the
fertilized eggs of a number of fish species, including carp, catfish, trout, salmon, arctic char,
and tilapia.
• The pronuclei of fish are not readily seen under a microscope after fertilization; therefore,
linearized transgene DNA is microinjected into the cytoplasm of either fertilized eggs or
embryos that have reached the four-cell stage of development.
• Unlike mammalian embryogenesis, fish egg development is external; hence, there is no need
for an implantation procedure.
• Development of transgenic fish occurs in temperature-regulated holding tanks.

39. • The survival of fish embryos after DNA microinjection is high (35 to 80%), and the
production of transgenic fish ranges from 10 to 70%.
• The presence of a transgene is scored by PCR analysis of either nucleated erythrocytes or
scale DNA.
• One of the most important aspects between fish and other terrestrial animals for cultivation
and genetic improvement is that, usually, fishes have higher levels of genetic variation and
hence more scopes for selection than most mammals or birds
The following are the important points needed for genetic engineering (gene transfer) to
produce transgenic fish:
(1) A gene sequence is to isolate for the particular characteristics; for example, growth
hormone gene.
(2) These genes (gene sequence) are then inserted into a circular DNA known as plasmid
Vector (enzymes endonucleases and ligases are used).
(3) Plasmids are harvested in the bacteria to produced billions of copies.
(4) Plasmids are introduced into linear DNA. The linear DNA is sometimes called a gene
cassette because it contains several sets of genetic material in addition to new inserted gene;
for example, growth hormone gene. The technology is available to integrate genes in germ line
of developing individual (fish) and finally transmitted into further generations.
(5) Making the cassette a permanent part of fish’s genetic makeup

40. • Transgenic salmon
• Natural salmon do not produce growth hormone in the cold, winter weather, and can take up
to 7 years (although usually 3 years) to reach reproductive age.
• Transgenic salmon consist of DNA from both the Pacific Chinook Salmon and an eel-like
fish, called the Ocean Pout, which allows it to keep pumping out growth hormone year-round
• With year-round growth hormone, the modified fish reach full size in less than half the time,
making it cheaper and more efficient for fish farms.

41. • Transgenic Tilapia:
• Tilapia fish, native to Africa, are cultured world-wide as “poor man’s food”
• Transgenic tilapia, which is modified with pig growth-hormone, has three times
larger than their non-transgenic siblings. Tilapia genetically modified with
human insulin grew faster than non-transgenic siblings, and could also serve as
a source of islet cells for transplantation to human subjects
• Transgenic Medaka Fish
• Inserting a gene construct consisting of the human growth hormone driven by the
salmon growth promoter into medaka produced the transgenic medaka.
• In another experiment Silk moth genes were introduced into Medaka fish to create
resistance to bacterial pathogens

42. • Transgenic Zebra Fish:
• The tiny zebra fish (Bmchydanio rerio) that lives in aquariums, was genetically
modified to produce a fluorescent red pigment, and is being promoted for sale as a
household aquarium pet, the “goldfish”.
• Gong (2003) developed novel varieties of the Zebra fish. Three “living
colour” fluorescent proteins, green fluorescent protein (GFP), Yellow fluorescent
protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a
strong muscle-specific mylz2 promoter in stable lines of transgenic zebra fish.
• These transgenic zebra fish with vivid fluorescent colours (green, yellow, red or
orange) fluorescent proteins can be seen with naked eyes under both daylight and
ultraviolet light in dark. The green fluorescent protein (GFP) is originally isolated
from the jellyfish (Aequorea tictoria)

43. Auto-Transgenesis:
Indian scientists are concentrating on developing transgenic fish through auto-
transgenesis which involves just increasing the copies of growth hormone genes
present in a fish as opposed to allotransgenesis which amounts to transfer of genes
from different species.
The increase in growth homone genes leads to an increase in flesh content. Indian
scientists feel that auto-transgenesis is safer and less controversial

44. • Disadvantages
• Low survival rate of transgenic animals.
• Can lead to mutagenesis and functional disorders
• For transgenic pigs, the pollutant phytase is discharged
• Transgenic sheep is a difficult and expensive procedure.
• Expensive
• some changes in environmental cycles (Insects not being able to eat their usual food and
needing to find new food sources)
• Is a lengthy proccess
• Advantages
• Allow scientists to study diseases in a more simple and efficient way Provide a way to
efficiently manufacture pharmaceutical products
• provide protein for human therapy. (Goats, Sheep, Chickens)
• For toxicologists, transgenic animals provide a more efficient subject to test for toxicants
because they are more responsive.
• Transgenic organisms have increased growth rates, improved disease resistance, increased
muscle mass, improved food conversion rates,improved nutritional quality, and improved
wool quality(sheep)
• Transgenic sheep allow scientists to study recombinant DNA.
• The use of transgenic animals, lowers the amount of experimental animals used during
testing. (less dogs and chimpanzees)
• Transgenic animals become better livestock(Sheep grow more wool, pigs grow more fat,
cows provide more milk)