By : Dr. Ashwini J. Patel

M. V. Sc Scholar (Animal Genetics & Breeding )

College of Vet. Sci. & A. H, Anand.

1) INTRODUCTION

2) HISTORY

3) WHY TRANSGENIC ANIMALS ?

4) STRETERGIES TO PRODUCE TRANSGENIC ANIMAL

5) METHODS / TECHNIQUE FOR PRODUCTION OF

TRANSGENIC ANIMALS

6) APPLICATION OF TRANSGENIC ANIMALS

1) INTRODUCTION

Transgenesis

The stable, one or more integration of foreign genes /foreign DNA into a host’s chromosomes.

or

Transgenesis either means transferring DNA into the animal or altering DNA of the animal.

Transgenic animal

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome .

or

Transgenic animal are genetically modified to contain a genefrom a different species following gene transplantation orresulting from the molecular manipulations of endogenousgenomic DNA

2) History

2) History:

Prior to the development of molecular genetics, the only wayof studying the regulation and function of mammalian genes wasthrough the observation of inherited characteristics orspontaneous mutations.

The discovery of DNA and genes opened wide avenues forresearch and biotechnological applications.

The introduction of isolated genes into cells became a commonpractice in the 1970s, soon after the emergence of the geneticengineering techniques.

During the 1970s, the first chimeric mice were produced.

It represented a great progress for the understanding of genefunction and mechanisms of action.

The first transgenic animal i.e. mice, were obtained bymicroinjecting the genes into one of the nuclei (pronuclei) ofone day old embryos.

U Pennsylvania University of WashingtonRalph Brinster Richard Palmiter

1982 , The First transgenic mouse with a phenotype

A team led by Ralph Brinster and Richard Palmiter made

a construct in which the rat growth-hormone gene was placed

under the control of zinc-inducible metallothionin promoter.

This construct was injected into fertilized mouse embryos andthe resulting transgenic offspring, were fed with extra zinc,which turned on the metallothionin promoter.

This resulted in the expression of growth hormone gene and theresulting high levels of circulating rat growth hormonedramatically changed the phenotype of the transgenic mice bystimulating them to grow twice as large as normal.

PALMITER, R.D., BRINSTER, R.L.,

HAMMER, R.E., TRUMBAUER, M.E.,

ROSENFELD, M.G., BIRNBERG, N.C.

and EVANS, R.M.

“Dramatic growth of mice that develop

from eggs microinjected with

metallothionin-growth hormone fusion

genes.”

Nature (1982) 300: 611-615.

• The giant mice instilled major excitement in the scientific and

public communities, markedly enhancing attention on the

transgenic mouse system.

• Ralph L. Brinster and Richard Palmiter thus were pioneered in

the development of methods to transfer foreign genes into the

germline of animals.

This technique is still widely used, gene transfer into animalsand plants to generate lines of genetically modifiedorganisms, known as transgenic animals and plants,respectively.

This method could be extrapolated (extend the application of toan unknown situation by assuming that existing trends willcontinue successfully) to other mammals in 1985.

Other transgenic animals include rats, pigs and sheep etc.

Transgenic technology led to the development of fish thatenabled to grow faster and livestock that enables to fightdiseases (prion-free cows resistant to bovine spongiformencephalopathy, known as mad cow disease).

Two other main techniques were Subsiquently developed:those of retrovirus-mediated transgenesis (Jaenisch, 1976)and embryonic stem (ES) cell-mediated gene transfer(Gossler et al., 1986).

The term transgenic was first used by J.W. Gordon and F.H.Ruddle (1981).

The transgenic technology also became an excellent tool inbasic research for understanding the functions andregulations of a number of mammalian genes.

Thanks to the transgenic technology, because today we have

mouse models for several types of cancer and of human

genetic disorders including chronic hepatitis, diabetes,

Alzheimer's disease and many more.

Transgenic mouse

3) Why Transgenic Animal?

Transgenic mice are often generated to :

1. characterize the ability of a promoter to direct tissue specific

gene expression

e.g. a promoter can be attached to a reporter gene such as

LacZ or GFP

2. examine the effects of over expressing and misexpressing

endogenous or foreign genes at specific times and locations

in the animals

Interest in transgenic animals originally fall into two broad

categories:

1. To increase production efficiency of farm animals in a short

duration.

2. Molecular farming: Using livestock to produce medicines,

nutraceuticals and tissues for transplant into humans.

4) Strategies for Producing Transgenic Animals

There are two basic strategies for producing transgenicanimals, which include “gain of function” or “loss offunction” transgenics.

The basic idea behind the gain of function strategy is that byadding a cloned fragment of DNA into an animal’s genome toa new gene product is produced that did not previouslyexisted in that cell or tissue. E.g. expression of rat growthhormone in mouse and to get over expression of gene productin the proper tissue (Palmiter et al., 1982).

The silencing or loss of gene function is accomplished by thetarget gene disruption through the process of homologousrecombination between host genome and exogenous DNA.

5) Techniques / Methods

of

Gene Transfer

5) Techniques/ Methods of Gene Transfer

• There are many techniques, all are listed below

1) Pronuclear injection

2) Transposon

3) Viral technique (Retroviral technique, Lenti

viral transfection)

4) ICSI / Sperm Mediated Gene Transfer

5) Embryonic stem cell / chimeras

6) somatic cell nuclear transfer / cloning

7) Chemical technique

8) Electroporation

9) RNA Interference

• Some of important methods have been described in Houdebine2003, 2005 and they are summarized in figure demonstated on next page.

Houdebine 2003, 2005

1) DNA Microinjection

About 1,000 copies of the isolated foreign gene contained in

1–2 pl may be injected into one of the pronuclei of one day

old mammalian embryos.

This method implies a superovulation of the females

followed by a mating with a male.

The resulting embryos are collected the next day and

microinjected with DNA. The embryos are then transferred

to hormonally prepared recipient females using surgery

operations.

The yield of this method in mice is of 1–2 of transgenics from

100 microinjected and transferred embryos. It is lower in all

the other mammalian species and very low in ruminants.

Superovulation and mating

Isolation of one cell stage zygote

Microinjection of transgene

Oviduct transfer to pseudopregnant females

Identification of founders by Southern blotting/PCR

In non mammalian species, the pronuclei cannot bevisualized and DNA must be injected into the cytoplasm ofthe one day old embryos. This relatively simple technique isefficient in most fish species.

Generation of sufficient number of eggs formicroinjection / superovularion.

Pregnant mare’s serum (or=FSH) on day 1

Human Chorionic Gonadotropin (or=LH) on day 3

Mated with a fertile male on day 3

Fertilized oocytes microinjected on day 4 with foreign DNAconstruct.

The microinjected eggs are implanted the same day or areincubated overnight and implanted into the oviduct ofpseudopregnant female the next day.

Mated female

Micro inject purified DNA into a malepronucleus of fertilized mouse egg.

Most eggs do not survive or do nothave the transgene, but between 1%and 30% of the eggs injected canproduce a live transgenic animal.

Generation of pseudopregnant females

Female mice are tricked into thinking they are pregnant

A female mouse in estrus is mated with a vasectomized male

Pseudopregnancy

If eggs (blastocysts) implanted will become truly pregnant and

will give birth to live offspring.

Vasectomy of male mice

~30 cells

200-250cells

2) Transformation of embryonic stem cells

Micropipette ES cells from the inner cell mass of a blastocyst

(i.e. early mouse embryo) in a strain with a physically

recognizable phenotype (e.g., pigmented).

Introduce transgenic construct/DNA into ES cells by

electroporation or viral vectors

Culture the cells in presence of antibiotic. Cells which are not

transgenic (black dots) will be killed, while those that have

taken up the DNA and are transgenic (red dots) survice.

• Insert the transgenic stem cells into the blatocyst of a mouse

with a different genetic background trait (e.g., an albino if the

original stem cells came from a pigmented mouse).

Implant the new blastocysts into a pseudopregnant

female with a visible phenotype different from the

blastocyst phenotype (e.g., albino if the blastocyst is

pigmented).

• Offspring that have pigmented sections are chimeras that

have incorporated the transgenic sequence into their cell lines.

Select them for further breeding.

• Keep breeding the offspring of the chimeras (two

heterozygotes) and their offspring will have all three

genotypes wild type homozygotes, heterozygotes, and

transgenic homozygotes(fully pigmented mice)

Black mouse -

no ES cell integration

Chimeric mouse -

high ES cell integration

Chimeric mouse -

low ES cell integration

Method : 2

Method : 1

3) DNA transfer via cloning:

The foreign gene is transferred into a somatic cell, the

nucleus of which is introduced into the cytoplasm of an

enucleated oocyte to generate a transgenic clone.

Cloning by nucleus transfer

Dolly“ 1997, first living offspring derived from

a differentiated cell.

5) Sperm Mediated Gene Transfer

The sperm cells have the capacity to bind naked DNA orbound to vesicles like liposomes (Lavitrano et al., 1989;Chang et al., 2002).

These sperm cells are in turn used for introducingexogenous DNA into oocytes either through invitrofertilization or artificial insemination.

Sperandio et al. (1996) successfully carried out the sperm

mediated gene transfer in cattle.

6) Retro viral technique Retrovirus is single stranded RNA virus which upon

transfection gets converted to double strand DNA andintegrates into the host genome (Eglitis et al., 1988).

The retroviral method was the first method to produce atransgenic mouse as reported by Jaenisch et al. (1975).

The most commonly used retroviral vector is Moloneymurine leukemia virus (Laneuville et al.,1988).

7) Lenti viral Transfection This method overcomes the limitations of viral mediated

gene transfer like low expression and silencing of genelocus.

Stable transgenic lines could be produced by injection thelentivirus into the perivitelline space of zygotes.(Hofmann et al., 2003).

8) Electroporation

This technique was developed by Puchalski and Fahl (1992).

In this technique, cells are exposed to electric field whichcauses the membranes to become polarized and a potentialdevelops across the membrane thereby breaking at localizedareas .

The cell becomes permeable to exogenous molecule.

The method has a greater efficiency either alone or incombination with other.

9) Chemical technique

This technique utilizes the chemical mediated uptake ofDNA or gene fragment by the host cell. The transfection iscarried out effectively by using chemicals like calciumphosphate or diethyl amino ethyl dextran.

10) RNA Interference

In this method, small interference RNAs (siRNAs), which

are 20-25 nucleotides long, bind to their complementary

sequences on target in mRNAs and shut down the

expression of genes and there by the production of protein

is stopped.

This RNA could be used for either transient or stable gene

repression or knock down of specific target genes.

Limitations of Transgenesis

The transgenic technology even though has tremendous

applications in livestock improvement programmes, still it has

lots of limitations:

Insertional mutations resulting in alteration of important

biological processes.

Unregulated gene expression resulting in improper expression

of gene products.

Possibility of side effects in transgenic animals like arthritis,

dermatitis and cancer etc.

Integration of exogenous DNA sequence in Y chromosome

resulting in transmission only to males.

Applications of

Transgenic Animals

1) Transpharmers

Transpharmers' are transgenic animals engineered to producetherapeutics, such as insulin in their milk, and used to treatdeficiencies such as diabetes

The proteins are produced in the mammary glands andharvested through the animals milk, The process can yieldbetween 1 and 10 grams of protein per liter of milk

A) Transpharmer Cattle-

Although cows are the most difficult animal to maketransgenic.

Herman the bull was genetically engineered to carry thehuman gene for the production of Lactoferin.

Human Lactoferin is a protein essential for the immunesystem of infants, and is present in mother’s milk, but does notnaturally occur in cows milk.

B) Transpharmer Goats

Transgenic dairy goats have been engineered to carry the

transgene for recombinant human antithrombin III (a blood

thinning protein).

Those produced 5g/L of recombinant anti-thrombin III protein

in their milk.

This drug marketed as ATryn® by Genzyme Transgenetics

Corp (GTC) became the world’s first FDA approved drug in

2009 (ATryn, 2009).

Three Transgenic Goats Expressing Recombinant Human Antithrombin Gene.

c) Transpharmer sheep [Tracy(1990-1997)]

The transpharmer sheep (ewe) was produced for production of

large amounts (< or = 65 grams per litre) of enzymatically

active human alpha 1 antitrypsin in the milk of transgenic

sheep which is used as potential treatment of cystic fibrosis.

It inhibits a wide variety of proteases and it protects tissues

from enzymes of inflammatory cells, especially neutrophil

elastase but the concentration can rise many fold upon acute

inflammation

In its absence, neutrophil elastase is free to break down

elastin, which contributes to the elasticity of the lungs,

resulting in respiratory complications such as emphysema, or

COPD (chronic obstructive pulmonary disease) in adults and

cirrhosis in adults or children.

2) Xenotransplanter pig

• Pigs produces a sugar alpha-1-galactose that the human body

recognizes as foreign and rejection of organ occur.

•The transgenic work attempted to knockout the genes

encoding the glycosyl-transferase enzymes that add the sugar

to organ surfaces, to counteract the rejection process

3) Transgenic Food Sources

Transgenic food sources aim to create animals that growlarger, mature quicker, are more nutritious and moreefficiently utilize the consumed food that sustains theanimal.

A) Super Fish

Coho salmon possessing the growth hormone (GH)transgene appear in market as the transgenic animal foodsources.

4) Scientific/Biological Models

There are marketable applications as for example giving ahousehold pet a greater communication capacity with theirowners.

Transgenic biological models have been used to increaseintelligence in mice, increase size in mice, and in one case toimpart the green fluorescent protein of a jellyfish in a monkey.

A) Super Mouse

This mouse received a rat growth hormone gene, and thetransgene was expressed to create very large mice relative totheir non-transgenic littermates.

B) Smart Mouse

One of the most significant scientific which ever successfully

conceived was the creation of a strain of smart mice named

Doogie.

These mice were endowed with the gene NR2B which is a

subunit of the glutamate receptor that predominates during

early development when learning and memory are easier.

This gene greatly increased a mouse's ability to recognize

objects, learn more effectively and increase spatial learning.

• Genetically engineered fluorescent

fish.

• zebra fishes were the first GloFish

available in pet stores.

• They are now sell in bright red,

green, orange-yellow, blue, and

purple and many more fluorescent

colors.

• It is one of the first genetically

modified animals to become publicly

available.

Glo fish.

5) Disease Models

One of the most important applications of transgenic

technology is the modeling of human diseases.

A) Huntington's Mouse

Huntington's disease (HD) is a neurodegenerative autosomal

disorder that typically onsets between 30 and 50 years old, but

can start at any age it is characterized by chlorea (involuntary

muscle spasms and loss of motor control)

Huntington's neural degeneration is caused by a mutation in the

huntingtin gene with CAG triplet nucleotide duplication,

which causes a build up of polyglutamine amino acids in

neurons.

A study in 1998 showed that mice expressing the mutated

human Huntington protein and also showed nuclear inclusion

bodies

This Huntington's disease model is just a minor sampling of

the scientific knowledge gained from transgenic mice. Other

models have also been constructed for Alzheimer’s disease,

cancer, AIDS, and Parkinson’s disease

B) Polio virus receptor

• Normal mice can't be infected with polio virus. They lack

the cell surface molecule that, in humans, serves as the

receptor for the virus.

• Transgenic mice expressing the human gene for the receptor

can be infected by polio virus and even develop paralysis

and other pathological changes characteristic of the disease in

humans.

• So, normal mice can serve as an inexpensive, easily

manipulated model for studying the disease.