BY

SONIA JAIN

BTB/12/316

• 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.

• 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.

• 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

STEP 1 :CONSTRUCTION OF A TRANSGENE

A transgene constitutes of 3 parts

• Promoter

• Gene to be expressed

• Termination sequence

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)

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.

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.

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

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).

(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

imperfect

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

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

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

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.

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

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.

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.

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.

THANK YOU!