Animal Biotechnology

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

Eggs are infected prior to fertilization Virus integrates into one of the chromosomes Recombinant Defective Retrovirus

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

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 Transgenic Animal Generation

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

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

The transgene used to increase growth utilizes an antifreeze protein promoter connected to the GH cDNA As water temperature drops the GH gene is turned on The fish continue to grow when normally they would not Antifreeze promoter from pout

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?

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 wild transgenic 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

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

Webster and Peter Nexia Biotechnologies transfered the silk gene from Orb spiders into goats The resulting male goats were used to sire silk-producing 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 goats 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

Other Types of Transgenic Animals

Transgene -> Gene coding for a growth hormone

ANDi, the first transgenic primate born in January, 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

Alba, the EGFP (enhanced GFP) bunny Created in 2000 as a transgenic artwork

Transgenic Pigs Pass on the Transgene

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

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 KOd 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

Blastocyst Injection BlastocystES cells

Chimeric mouse The brown fur comes from ES cells injected into the blastocyst of an albino mouse

Some Examples of Knockout Mice

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) p27 knockout mouse

GDF8 (Myostatin) knockout mouse Over twice the muscle mass of a wildtype mouse normalknockout

Naturally Occurring GDF8 Mutants

FGF5 knockout mouse has long, angora-like hair

Clones and Cloning

Dolly as a lamb with her surrogate mother Dolly, First Mammal Cloned From an Adult Cell Dolly, as an adult

Somatic Cell Nuclear Transfer

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

Cloned transgenic cat containing red fluorescent protein Transgenic Clones

Idaho Gem, first cloned mule 1 st try 134 implants 2 pregnancies, both failed 2 nd try 113 implantations 14 pregnancies, one birth Surrogate mother (horse)

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

Conservation Cloning Many endangered or extinct animals are being cloned or considered for cloning Gaur Bucardo mountain goat Mammoth Quagga Banteng

MammothQuagga BucardoGaur

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

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% Problems with Cloning

Nearly all clones show some genetic anomalies Some suffer from placental defects others cardiac defects Many suffer from large offspring syndrome (LOS) Normal mouse pupCloned mouse pup suffering from LOS