Transgenic animals

Hoza, A.S

BLS 209 Lecture

What is a transgenic animal?

• An animal contains a foreign gene (genes)

introduced purposely by human intervention

•Transgenic animals are altered so that their DNA

produce proteins that normally they would not

produce

History of transgenic animal production

1970's, first transgenic mice via viral

infection, but not germline transmission

1980's, first transgenic mice via microinjection,

the most popular technique

1985, first transgenic rabbits, sheep, pigs and

cattle

80-90, commercial transgenic services, via

transgenic facility

1990's, transgenic farm animal companies as

bioreactors and organ donors

Different ways to create transgenic animals

Target gene – transgene

Promoter/enhancer - when, where, how much

Coding sequence - coding the specific protein

ploy A detail - mRNA stability

Transgene Structure

Gene transfer methods

1. Microinjection of recombinant DNA into the male

pronucleus of an in vitro fertilized egg.

2. Embryonic stem cell transfer (ES).

Other methods:

1.Chemical or

2. Viral delivery into ES cells, or homologous

recombination with ES cells.

Microinjection

• Inject DNA molecules (transgenes) directly into

male pronucleus

• Most popular technology, commercial available

• Success rates range from 10-30% depending on

skills and constructs

•Efficiency is not related to the copies of transgenes

injected

Microinjection

• The technique can be applied to other species

• No theoretical limit for the size of the construct

• Overall efficiency is still low, particularly for farm

animals

• Tandem repeat of gene constructs (head-tail)

• High frequency of mosaic

• Initial investment is high

Virus mediated gene transfer

• Earliest method for successful gene transfer in

mammals

• Virus has transfection property

• Killed virus is replication defective

• The virus gene is replaced with transgene gene

• The transgene is delivered to the host cell by

transfection (gene therapy)

• Can be used to transfect a wide range of cells, e.g.,

ES cells

Virus mediated gene transfer

•Direct transfection of embryos has resulted

nongermline transgenics

• ES cells transfection has resulted in germline

transgenics

• Has succeeded in chickens and fish

• Transfecting oocytes resulted in 100% transgenics

• Only small transgene construct is usable (8 kb or

less)

• More research is needed on the safety of the

method

Embryonic stem cells

•Used mostly when trying to target a transgene to

a specific site in the genome.

• Derived from ICM of blastocyst stage embryo

• Divide in vitro indefinitely without differentiation

• Contribute to development of the fetus in any

tissues, organs (germline)

• Has the potential to give rise to all tissues

ES cells

• May be transfected with transgene or with

genes removed (knockout) or inserted prior to

microinjection

• Has revolutionized genetics, development,

immunology and cancer research in mice

Steps for ES Cell Manipulation.

Approaches to using ES cells to create transgenic

animals.

1. The transgene can be microinjected into the

ES cells

2. can be introduced by a virus,

3. Chemical (e.g calcium phosphate or rubidium

chloride

4. by using homologous recombination.

Nuclear transfer

• Creation of Dolly

• Somatic cells be transfected, or genetically altered

prior to NT

• 100% efficiency of any progeny

• Low efficiency

• Abnormal development

Screening for Transgenic Positives

• Identification of transgene integration - DNA

• Detect transgene transcription - mRNA

• Detect transgene expression - protein

TRANSGENIC ANIMAL CLASSIFICATION

Five major categories:

1. disease models

2. transpharmers

3. xenoplanters

4. food sources

5. scientific models

1.Disease models:

animals that have been modified to exhibit the

symptoms and progression of a particular disease,

so that treatments for that disease can be tested

on them (e.g oncomouse, AIDS mouse etc)

2.Transpharmers:

animals modified to express a particular

protein or suite of proteins in their milk to avoid

animal sacrifice when obtaining the drug.

The proteins can be purified to produce

medicines and hormones to treat humans, or

can possibly be administered as medicinal milk

itself.

Mice- commonly used to test the transpharming

transgene first.

The transgenic procedure is promising, but very

expensive, and still has a low success rate especially

for larger farm animals.

A mouse engineered in 1987 to express the clot

dissolver drug tissue plasminogen activator (tPA)

In 1990, human alphaantitrypsin, an inhibitor used to

treat emphysema, was produced in the mouse’s milk.

1997 at the New Technology Institute- human alpha-

lactalbumin in mouse’s milk

Larger animals like sheep, goats, and cows are the targets

for large-scale transpharming.

E.g 6 transgenic lambs for Roslin Institute - created in

1997 to produce a human clotting factor in their milk.

The first transpharmer goats were produced in 1991 at

the Tufts University School of Veterinary Medicine to

produce tissue plasminogen activator, a clotdissolving

drug.

transpharmer goats were produced in 1999 using

SCNT contained high levels of human antithrombin

III.

The first transgenic cow (Gen Pharm Intern, California),

dubbed “Herman”, and his first transgenic offspring were

bred at Gen Pharm’s lab (Netherlands)

Two calves were produced by microinjection of DNA into

embryos that were then implanted in surrogate mothers and

born alive.

One of these cows was female the transgene

rearranged itself so that a portion of the lactoferrin cDNA was

deleted.

The other calf was male, later called “Herman.” He and his

offspring contained the correctly arranged gene for human

lactoferrin.

4. Food sources:

animals that grow bigger or faster to produce

more food in a shorter amount of time with fewer

resources. E.g superpig, superfish

3. Xenoplanters:

animals that have been engineered to not

express the foreign antigens that normally

prevent the transplantation of their organs into

humans.

5. Scientific models:

animals producing more or less of a

particular protein than usual,

Study that protein’s purpose in biological

mechanisms or development applied tohumans. E.g ANDi first transgenic monkey

TRANSGENIC ETHICS

1. Animal Rights Versus Animal Welfare

2. Right to meddle in the genomes of living beings

Transgenesis- a logical step beyond selective

breeding,

open doors past what we previously have

known to cure diseases!!??

possibly end world hunger entirely!!!??

Transgenic Art - Creating monsters!!! E.g “Alba,” the rabbit that glows under UV light!!!!???

Eduardo “transgenic art.” refers to animals and plants with a

planned genome intended to express an artistic idea

symbolized by the proteins they code for.

4. Animal Death Versus Human Lives Saved

low success rate in creating transgenic animals.

5. Transgenic Animals and the Environmentdecrease of genetic variability within that species

Transgenic animals are not “more fit” than their “normal” cousins.

6. Transgenic Oversight

Transgenic experimentation should be as

humane as possible.

7. Religions and Transgenic Ethics

Applications of transgenic farm animals

• Agricultural applications

• Bioreactors

• Organ/cell/tissue donors

• Basic research/disease model