Transgenic pigs have been developed through inserting foreign DNA into pig genomes using various techniques. Pigs are useful biomedical models because their physiology is similar to humans. Transgenic pigs have been created for various purposes, such as producing human proteins in their milk or blood, modeling human diseases, producing organs for xenotransplantation, and reducing phosphorus pollution through modified digestion of phytates. Genetic engineering of pigs continues to be studied for applications in biomedicine and agriculture.

1. BY
SONIA JAIN

2. • Transgenic animals are the animals with modified genomes
containing foreign DNA
• Transgenesis is the process by which mixing up of genes
takes place
• Transgenic technology has led to the development of
fishes, live stock and other animals with altered genetic
profiles which are useful to mankind.
• First transgenic animal was a ‘Supermouse’ created by
Ralph Brinster (U Pennsylvania) and Richard Palmiter
(University of Washington) in 1982.

3. • Rodents are the most favored animals used as models because of
their short generation interval, size, availability, and housing cost.
• However, there is a distinctive difference in physiology between
humans and rodents which obstruct the direct application of
knowledge to human medicine.
• 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,
pathobiology of intestinal transport, injury and repair, repair and
healing of wounds, etc.

4. • Also been considered for being potential source of different
organs for the xenotransplantation as can be seen in the heart
transplantation studies
• 1985 First transgenic pigs by Microinjection of DNA into one
pronucleus of a zygote

5. STEP 1 :CONSTRUCTION OF A TRANSGENE
A transgene constitutes of 3 parts
• Promoter
• Gene to be expressed
• Termination sequence

6. STEP 2:INTRODUCTION OF FOREIGN GENE
There are a number of methods to carry out the genetic modification of
the animals
a) 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. This technique is commonly used to study the role of genes
using mouse animal models .In order for pronuclear injection to be
successful, the genetic material (typically linear DNA) must be
injected while the genetic material from the oocyte and sperm are
separate (i.e., the pronuclear phase)

8. b) retrovirus mediated transfer
In this method the genes are removed from the genome of lentiviruses (a
category of retroviruses) and replaced by the genes of interest.
This is integrated into the chromosomes of the oocyte after its injection
between the zona pellucida and the plasma membrane of oocyte that is
arrested in the metaphase II of meiosis.
It was carried out in pigs after application in cattles.
Immediately following infection, the retrovirus produces a DNA copy of
its RNA genome using its reverse transcriptase.
The DNA copy of the viral genome, or provirus, integrates randomly into
the host cell genome, usually without deletions or rearrangements.

10. c) 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.

11. d) somatic cell nuclear
transfer (SCNT)
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

12. As targeted integration is not achieved properly in other methods, it
makes way for the development of Embryonic stem cells (ES cell
technology).
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, a true ES cell that goes with germline is yet to be
developed.
(e) Embryonic stem cell technology (ES cell technology).

14. (f) Zinc finger nucleases
Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a
zinc finger DNA-binding domain to a DNA-cleavage domain.
Zinc finger domains can be engineered to target desired DNA sequences and this
enables zinc-finger nucleases to target unique sequences within complex genomes. By
taking advantage of endogenous DNA repair machinery, these reagents can be used to
precisely alter the genomes of higher organisms.
A zinc finger is a small protein structural motif that is characterized by the
coordination of one or more zinc ions in order to stabilize the fold
ZFNs can be used to produce double-strand breaks (DSBs) in the DNA (see Genetic
recombination) in the mutant allele, which will, in the absence of a homologous
template, be repaired by non-homologous end-joining (NHEJ). NHEJ repairs DSBs by
joining the two ends together and usually produces no mutations, provided that the
cut is clean and uncomplicated. In some instances, however, the repair will be

15. The introduction of genetic modification using zinc finger nucleases in
combination with the donor stem cells may prove to be a highly efficient
method for the genetic modification of swine

16. Analysis techniques
• PCR technique
• Analysis of transgene integration
• Analysis of mRNA production
• Analysis of protein expression
• selectable marker is used to differentiate
transformed from untransformed cells

17. 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

18. 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.

19. 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

20. 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.

22. The different applications of genetically modified pigs in medical field can be summarized as
follows:
1) 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.
2) 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.

23. 3) 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.
4) Production of human and pig hybrid organs is a very interesting application that
needs further in-depth study. 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.
5) Transgenic porcine livers expressing albumin gene are being studied for use as
bio-artificial liver support system as a bridge to human liver transplantation.
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.

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