Animal Biotechnology

Animals in Research
 Animals in Research

Animation of
Drug
Development
Process
 Animals in Research
❚ Animal Models
• Mice
• Rats
• Zebrafish (3 month generation time,
200 progeny, complete embryogenesis
in 120 hrs)
• Dogs (lungs and cardiovascular
system)
• Cats
• Pigs (PPL Therapeutics- delete a gene
which causes hyperacute rejection of
pig-to-human organ transplantation)
• Primates (HIV and AIDs research,
geriatric research)
 Animals in Research

❚ Alternatives to Animal Models

• Cell culture devices
• Researchers use cell cultures and computer-generated
models whenever possible, but this doesn’t work for looking
at an organ or entire animal
 Animals in Research

❚ Regulation of Animal Research

• The “Three Rs”
• Reduce the number of higher species (cats, dogs,
primates) used
• Replace animals with alternative models whenever
possible
• Refine tests and experiments to ensure the most
humane conditions possible
 Animals in Research

❚ Veterinary Medicine as Clinical Trials

• Treatments for humans may also be useful for
treatments with animals (e.g. the BRCA1 gene found
in 65% of human breast tumors is similar to the
BRCA1 gene in dogs)
• Hyperthermia + radiation = more effective at killing
tumors
• Stimulation of cytokines for curing skin cancers
Transgenics are genetically modified organisms with DNA
from another source inserted into their genome
A large number of transgenic animals have been created
Mice Cows Pigs Sheep Goats Fish Frogs Insects
Currently, no transgenic animal or animal product is
approved by the FDA or USDA for human consumption
Some of the goals of transgenic animal creation are:
•Research into animal and human disease
•Improve livestock animals
•Use of animals as bioreactors
Transgenic Animal Creation
 Microinjection
into the germ line -> transgenic animal

Gene injected into the male

pronuclei
Recombinant Defective Retrovirus

Eggs are infected prior to fertilization

Virus integrates into one of the chromosomes
Linker Based Sperm-Mediated Gene Transfer
(LB-SMGT)

Sperm fertilizes the egg carrying the foreign gene

into the egg where it is incorporated into the genome
 Transgenic Animal Generation

Some of the drawbacks of these methods are:

•The inserted DNA randomly integrates into the genome
•The eggs must be harvested & fertilized in vitro
•More than one copy of the gene may get into the genome
Examples of Transgenic Animals
 Transgenic Cattle

Dairy cows carrying extra copies of two types of casein

genes produce 13% more milk protein
Not only will this make the milk more nutritious, it would
allow for less milk to make more cheese
Currently the milk from these animals is under FDA review
The important difference between this & other transgenics
is that the DNA added is not foreign
 EnviroPig TM

Transgenic pigs express phytase in their salivary glands

Phytic acid in the pig meal is degraded releasing phosphorus
The phosphorus is absorbed by the pig
Normally the phytic acid/phosphorus complex passes
through the pig and is excreted as waste
Pig waste is a major pollutant & can cause eutrophication of
lakes & streams

http://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg
 Transgenic Fish
Tilapia
Salmon/trout
Catfish
Can grow up to 6 times faster than wildtype fish
Most have extra copies of growth hormone (GH) gene

Transgenic
Wildtype

http://www.nature.com/nbt/journal/v19/n6/images/nbt0601_500a_I1.jpg
The transgene used to increase growth utilizes an
antifreeze protein promoter connected to the GH cDNA

Antifreeze promoter
from pout

As water temperature drops the GH gene is turned on

The fish continue to grow when normally they would not

http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
Concerns if these supersized transgenic fish got loose
Transgenic fish are farm-raised, isolated from wild stocks
But even during farming of wildtype fish, escapes happen
frequently (~14 million/yr)
What would happen if a large number of transgenic escaped
& started breeding with wild fish?

http://pewagbiotech.org/buzz/index.php3?IssueID=10
In experiments, transgenic males mated 3x more
frequently than the smaller wild males
Offspring of transgenic males lived <70% as long as
wild males
Could lead to a decline of the wild fish population &
endanger a species as whole
 Antifreeze Proteins (AFP)
AFPs lower the freezing temperature of blood & fluids
Trout normally do not survive in water below –0.6 C
Transgenic trout containing an AFP gene & promoter can
survive in waters as cold as –1.2 C

wild
transgenic +Antifreeze

http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
Animal Bioreactors
Pharming
1997, Tracy the sheep, the first transgenic animal to
produce a recombinant protein drug in her milk
alpha-1-antitrypsin (AAT) treatment for emphysema &
cystic fibrosis
Created by PPL Therapeutics & The Roslin Institute

http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml
Nexia Biotechnologies transfered the silk gene from Orb
spiders into goats
The resulting male goats were used to sire silk-producing
Webster and Peter

female goats
Each goat produces several grams of silk protein in her
milk
The silk is extracted, dried to a white powder, and spun
into fibers
The fibers are stronger and more flexible than steel

Transgenic male kids

carrying silk gene
GTC Biotherapeutics has received approval to sell human
anti-thrombin (ATryn) purified from goat s milk in Europe
Technology is not restricted to cows, goats, & sheep
There is interest in using rabbits since housing costs are
significantly less & generation time is faster
Chickens which produce recombinant drugs in their eggs
have been produced by The Roslin Institute
 Producing Human Antibodies in Animals

• Production of Monoclonal
antibodies (Mabs)
• Used to treat cancer, heart
disease, and transplant
rejection
• HUMANIZED monoclonal
antibodies were developed to
prevent the human anti-mouse
antibody (HAMA) response
Other Types of Transgenic Animals
Transgene ->
Gene coding
for a growth
hormone
ANDi, the first transgenic primate born in January, 2000
224 unfertilized rhesus eggs were infected with a GFP virus
~Half of the fertilized eggs grew and divided
40 were implanted into twenty surrogate mothers
five males were born,two were stillborn
ANDi was the only live monkey carrying the GFP gene

http://www.ohsu.edu/unparchive/2001/011001andi.shtml
Alba, the EGFP (enhanced GFP) bunny
Created in 2000 as a transgenic artwork

http://www.ekac.org/gfpbunny.html#gfpbunnyanchor
Transgenic Pigs Pass on the Transgene

http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
GloFish, originally developed in Singapore as a way to monitor
water pollution
The normally black-and-silver zebrafish was turned green or
red by inserting various versions of the GFP gene
Glofish are on sale throughout the US except in California
Glofish retail for about $5 per fish. Normal zebrafish cost
around one tenth of the price

http://www.nus.edu.sg/corporate/research/gallery/research12.htm
Mouse Knock-out Technology
Gene Targeting
Knock-out technology allows for the specific loss of a gene
in mice
Allows for the function of the KO d gene to be deduced
from the defects seen in the mice
can be used to mimick some disease
Unlike traditional transgenics the trangene is targeted to a
specific site in the DNA of the mouse
Mouse Knock-outs require embryonic stem (ES) cells
These are derived from the inner cell mass (ICM) of a
blastocyst (the ICM is what will become the fetus)
ES cells are pluripotent meaning they can become all the
different cell types found in an adult

http://cba.musc.edu/SC_COBRE/CORE-B/Resources-B.htm
 Transgenic Animals
❚ Knock-outs: A Special Case of
Transgenesis
• A specific gene is disrupted or
removed such that it is not expressed
• Procedure: DNA is modified, it is
added to embryonic stem cells,
where it undergoes homologous
recombination. The modified ES cells
are then introduced into normal
embryo. The embryo is implanted in
an incubator mother. The offspring is
a chimera. It may take several
generations of crossbreeding are
required to produce animals that are
complete knock-outs.
• Breast cancer mouse
Blastocyst Injection

Blastocyst ES cells

http://tasq.uq.edu.au/blasto.html
 Chimeric mouse
The brown fur comes from ES cells injected
into the blastocyst of an albino mouse

http://bunseiserver.pharm.hokudai.ac.jp/gihou/knockout.html
Some Examples of Knockout Mice
 p27 knockout mouse

p27 knockout mouse is bigger than the control

This is not due to obesity, but the skeletal structure is
increased in size (everything about the mouse is larger)

http://www.bioreg.kyushu-u.ac.jp/saibouE.html
 normal knockout

GDF8 (Myostatin) knockout mouse

Over twice the muscle mass of a wildtype mouse

http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
Naturally Occurring GDF8 Mutants

http://www.canada.com/victoriatimescolonist/story.html?id=67f15c17-2717-4022-bb76-1b982456e793&k=94653
http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
FGF5 knockout mouse has long, angora-like hair

http://www.med.uni-jena.de/ivm/deutsch/method/method_7.htm
Clones and Cloning
 Dolly, First Mammal Cloned From an Adult Cell

Dolly, as an adult
Dolly as a lamb with
her surrogate mother

http://www.harlemlive.org/community/health-science/scientificcommunity/index2.html
 Somatic Cell
Nuclear Transfer

http://www.howstuffworks.com/cloning3.htm
 What Has Been Cloned So Far?

Somatic Cell Nuclear Transfer

Sheep, Goat, Mouse, Rabbit, Cattle (domestic & wild),
Pig, Horse, Mule, Dog, Cat (domestic & wild), Deer

Embryo Splitting (Twinning)

Sheep, Cattle, Primate (Rhesus)
 Cat Clone

Donor Surrogate mother with clone (CC)

Out of 87 implants only CC survived to birth

Donor & Clone

Rainbow & CC

http://www.usatoday.com/news/science/2003-01-21-cloned-cats_x.htm
 Transgenic Clones

Cloned transgenic cat containing red fluorescent protein

http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
 Idaho Gem, first cloned mule

Surrogate
mother
(horse)

1st try 134 implants 2 pregnancies, both failed

2nd try 113 implantations 14 pregnancies, one birth

http://news.nationalgeographic.com/news/2003/05/0529_030529_muleclone.html
In addition to cloning pets or prized livestock,
researchers are looking to clone transgenic animals
This would allow for more uniform expression of
transgenic genes
Not all transgenic animals express their transgenic
genes at equal levels
Also allows for the rapid expansion to large flocks
or herds of transgenic animals
Piglets clones created by PPL Therapeutics in 2000
The piglets carry a silenced copy of alpha 1,3 galactosyl
transferase, or GT, an enzyme involved in organ rejection
In order to guarantee compatibility a second GT gene must
also be silenced

http://www.newrivervalley.com/biotech/day1.html
 Conservation Cloning
Many endangered or extinct animals are being cloned or
considered for cloning
Gaur
Bucardo mountain goat
Mammoth
Quagga
Banteng
Mammoth Quagga
Bucardo Gaur

http://www.personal.psu.edu/faculty/t/r/trp2/mammoth.jpeg
http://www.serragaucha.com.br/rocky/zoo.html
http://www.riosmith.net/Gaur004.jpg
http://www1.ceit.es/Asignaturas/Ecologia/EspNaturales/Ordesa/mamiferos.htm#Bucardo
Noah, a Banteng clone created by Advanced Cell Technologies
Banteng are endangered wild bovine from Southeast Asian
This clone was created from frozen tissue of an animal that
died in 1980

http://www.advancedcell.com/images/Banteng002-sm.jpg
 Problems with Cloning
3 Pig clones, born in 2002, died of heart attacks due to adult
clone sudden death syndrome within days of each other by
the time they were 6 months old.
Dolly had a weight problem, telomeres 20% shorter than
normal, she suffered from arthritis, and finally lung cancer
due to an infection for which she was finally euthanized at
age 6yrs.
The success rate ranges from 1 to 3% this contrasts to in
vitro fertilization which has a success rate of 50 to 20%
http://fig.cox.miami.edu/~cmallery/150/gene/cloning.success.jpg
Nearly all clones show some genetic anomalies
Some suffer from placental defects others cardiac defects
Many suffer from large offspring syndrome (LOS)

Normal mouse pup Cloned mouse pup

suffering from LOS
CONTOH HEWAN GMO
• Angelfish (Pterophyllum) yang dimodifikasi secara genetik
bersinar dalam akuarium di Taiwan. Ikan ini adalah angelfish
pertama yang memancarkan fluoresen pink dan bisa terlihat
dalam kegelapan.
Ikan Convict Cichlids (Amatitlania nigrofasciata) tampak bersinar
dalam tas plastik dalam sebuah akuarium di Taipei
Ikan Archocentrus nigrofasciatus bersinar dalam akuarium di
Taiwan
Dua babi transgenik terlihat menunjukkan protein fluorsen hijau
di kuku-kukunya di Harbin, Provinsi Heilongjiang, Cina
Dua ayam tanpa bulu di Hebrew University di Rehovot, Israel. Ayam ini rendah
lemak, ramah lingkungan karena mengurangi biaya yang harus dikeluarkan
peternak untuk ventilasi.
Noori (1,3 kg) lahir pada 9 Maret 2012 adalah kambing Pashmina pertama
hasil kloning, memiliki bulu yang hangat, hidup di padang rumput dengan
suhu udara yang bisa mencapai – 20 º C
Snuppy (kanan), anjing jantan pertama yang merupakan hasil kloning sel
dewasa dan seekor anjing tipe Afghan hound yang selnya diambil untuk
mengkloning Snuppy (Seoul)
Domba GMO - glow in the dark, lahir April 2013 di Uruguay