Слайд 2Transgenics 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
Слайд 3Some of the goals of transgenic animal creation are:
Research into
animal and human disease
Improve livestock animals
Use of animals as
bioreactors
Слайд 5Microinjection
into the germ line -> transgenic animal
Gene injected
into the male pronuclei
Слайд 6Eggs are infected prior to fertilization
Virus integrates into one of
the chromosomes
Recombinant Defective Retrovirus
Слайд 7Linker 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
Слайд 8Some 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
Слайд 9Examples of Transgenic Animals
Слайд 10Transgenic 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
Слайд 11EnviroPig 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
Слайд 12Transgenic 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
Слайд 13The transgene used to increase growth utilizes an antifreeze protein
promoter connected to the GH cDNA
http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
As water temperature drops the
GH gene is turned on
The fish continue to grow when normally they would not
Antifreeze promoter from pout
Слайд 14Concerns 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
Слайд 15In experiments, transgenic males mated 3x more frequently than the
smaller wild males
Offspring of transgenic males lived
as wild males
Could lead to a decline of the wild fish population & endanger a species as whole
Слайд 16http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
+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
Слайд 18http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml
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
Слайд 19Nexia 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
Слайд 20GTC 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
Слайд 21Other Types of Transgenic Animals
Слайд 22Transgene -> Gene coding for a growth hormone
Слайд 23ANDi, 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
Слайд 24Alba, the EGFP (enhanced GFP) bunny
Created in 2000 as a
transgenic artwork
http://www.ekac.org/gfpbunny.html#gfpbunnyanchor
Слайд 25http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Pigs Pass on the Transgene
Слайд 26GloFish, 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
Слайд 27Mouse “Knock-out” Technology
Gene Targeting
Слайд 28Knock-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
Слайд 29http://cba.musc.edu/SC_COBRE/CORE-B/Resources-B.htm
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
Слайд 31http://tasq.uq.edu.au/blasto.html
Blastocyst Injection
Blastocyst ES cells
Слайд 32http://bunseiserver.pharm.hokudai.ac.jp/gihou/knockout.html
Chimeric mouse
The brown fur comes from ES cells injected into
the blastocyst of an albino mouse
Слайд 34p27 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
p27 knockout mouse
Слайд 35http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
GDF8 (Myostatin) knockout mouse
Over twice the muscle mass of a
wildtype mouse
normal knockout
Слайд 36Naturally 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
Слайд 37FGF5 knockout mouse has long, angora-like hair
http://www.med.uni-jena.de/ivm/deutsch/method/method_7.htm
Слайд 39http://www.harlemlive.org/community/health-science/scientificcommunity/index2.html
Dolly as a lamb with her surrogate mother
Dolly, First Mammal
Cloned From an Adult Cell
Dolly, as an adult
Слайд 40http://www.howstuffworks.com/cloning3.htm
Somatic Cell Nuclear Transfer
Слайд 41What 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)
Слайд 42Cat Clone
Donor Surrogate mother with clone (CC)
Out of
87 implants only CC survived to birth
Слайд 43http://www.usatoday.com/news/science/2003-01-21-cloned-cats_x.htm
Donor & Clone
Rainbow & CC
Слайд 44Cloned transgenic cat containing red fluorescent protein
http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Clones
Слайд 45http://news.nationalgeographic.com/news/2003/05/0529_030529_muleclone.html
Idaho Gem, first cloned mule
1st try 134 implants 2 pregnancies,
both failed
2nd try 113 implantations 14 pregnancies, one birth
Surrogate mother
(horse)
Слайд 46In 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
Слайд 47http://www.newrivervalley.com/biotech/day1.html
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
Слайд 48Conservation Cloning
Many endangered or extinct animals are being cloned or
considered for cloning
Gaur
Bucardo mountain goat
Mammoth
Quagga
Banteng
Слайд 49Mammoth 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
Слайд 50http://www.advancedcell.com/images/Banteng002-sm.jpg
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
Слайд 513 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
Слайд 52http://fig.cox.miami.edu/~cmallery/150/gene/cloning.success.jpg
Слайд 53Nearly 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
