Cloning genes into animals Transgenic animal carries foreign gene inserted into its genome.
Chapter 7: Animal Biotechnology - WordPress.com€¦ · Currently, no transgenic animal or animal...
Transcript of Chapter 7: Animal Biotechnology - WordPress.com€¦ · Currently, no transgenic animal or animal...
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ANIMAL BIOTECHNOLOGY
ENDRIKA WIDYASTUTI
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Transgenics are genetically modified organisms with DNAfrom another source inserted into their genomeA large number of transgenic animals have been createdMice Cows Pigs Sheep Goats Fish Frogs InsectsCurrently, no transgenic animal or animal product isapproved by the FDA or USDA for human consumption
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Some of the goals of transgenic animal creation are:•Research into animal and human disease•Improve livestock animals•Use of animals as bioreactors
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Animals in Research
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Animals in Research
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Animals in Research
Animation ofDrugDevelopmentProcess
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z Animal Models• Mice• Rats• Zebrafish (3 month generation time,
200 progeny, complete embryogenesisin 120 hrs)
• Dogs (lungs and cardiovascularsystem)
• Cats• Pigs (PPL Therapeutics- delete a gene
which causes hyperacute rejection ofpig-to-human organ transplantation)
• Primates (HIV and AIDs research,geriatric research)
Animals in Research
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z Alternatives to Animal Models• Cell culture devices
• Researchers use cell cultures and computer-generatedmodels whenever possible, but this doesn’t work for lookingat an organ or entire animal
Animals in Research
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z Regulation of Animal Research• The “Three Rs”
• Reduce the number of higher species (cats, dogs,primates) used
• Replace animals with alternative models wheneverpossible
• Refine tests and experiments to ensure the mosthumane conditions possible
Animals in Research
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z Veterinary Medicine as Clinical Trials• Treatments for humans may also be useful for
treatments with animals (e.g. the BRCA1 gene foundin 65% of human breast tumors is similar to theBRCA1 gene in dogs)
• Hyperthermia + radiation = more effective at killingtumors
• Stimulation of cytokines for curing skin cancers
Animals in Research
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z Bioengineering Mosquitoes to Prevent Malaria• Cloned in a gene that prevents the parasite from
traversing the midgut; blocking the continuation of itslife cycle
• Developed an antibody that prevents the parasitefrom entering the mosquito’s salivary gland
Animals in Research
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Clones
z Cloning• Creating Dolly• Limits to Cloning: The donor cell must
come from a living organism• An organism is also shaped by its
environment• The success rate for cloning is very low• Clones may be old before their time
• The future of cloning: preservation ofendangered animals, studying the effectof drugs etc on duplicates, improveagricultural production
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Transgenic Animals
z Retrovirus-mediated transgenesisz Pronuclear microinjectionz Embronyic stem cell methodz Sperm-mediated transfer
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z Improving Agricultural Productswith Transgenics• Faster growth rates or leaner
growth patterns (improve theproduct), more product
• Increase nutritional content-lactoferrin
• Turning the animals into efficientgrazers
• Transfer antimicrobial genes tofarm animals
Transgenic Animals
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z Transgenic Animals as Bioreactors• Biosteel otherwise known as spider silk, cloned into
goat milk (“silkmilk” goats)• Goats reproduce faster than cows and are cheaper than
cows• Hens also make good bioreactors in that they are cheap
and a lot of eggs are produced at one time
Transgenic Animals
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z Knock-outs: A Special Case ofTransgenesis• 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 homologousrecombination. The modified ES cellsare then introduced into normalembryo. The embryo is implanted inan incubator mother. The offspring isa chimera. It may take severalgenerations of crossbreeding arerequired to produce animals that arecomplete knock-outs.
• Breast cancer mouse
Transgenic Animals
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Producing Human Antibodies inAnimals
z Production of Monoclonalantibodies (Mabs)• Used to treat cancer, heart
disease, and transplantrejection
• HUMANIZED monoclonalantibodies were developedto prevent the human anti-mouse antibody (HAMA)response
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Transgenic Animal Creation
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Microinjectioninto the germ line -> transgenic animal
Gene injected into the malepronuclei
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Eggs are infected prior to fertilizationVirus integrates into one of the chromosomes
Recombinant Defective Retrovirus
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Linker Based Sperm-Mediated Gene Transfer(LB-SMGT)
Sperm fertilizes the egg carrying the foreign geneinto the egg where it is incorporated into the genome
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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
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Examples of Transgenic Animals
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Transgenic Cattle
Dairy cows carrying extra copies of two types of caseingenes produce 13% more milk proteinNot only will this make the milk more nutritious, it wouldallow for less milk to make more cheeseCurrently the milk from these animals is under FDA reviewThe important difference between this & other transgenicsis that the DNA added is not foreign
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EnviroPig TM
Transgenic pigs express phytase in their salivary glandsPhytic acid in the pig meal is degraded releasing phosphorusThe phosphorus is absorbed by the pigNormally the phytic acid/phosphorus complex passesthrough the pig and is excreted as wastePig waste is a major pollutant & can cause eutrophication oflakes & streams
http://www.nature.com/news/2005/050221/images/nbt0305-283-I1.jpg
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Transgenic FishTilapiaSalmon/troutCatfishCan grow up to 6 times faster than wildtype fishMost have extra copies of growth hormone (GH) gene
TransgenicWildtype
http://www.nature.com/nbt/journal/v19/n6/images/nbt0601_500a_I1.jpg
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The transgene used to increase growth utilizes anantifreeze 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 onThe fish continue to grow when normally they would not
Antifreeze promoterfrom pout
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Concerns if these ‘supersized’ transgenic fish got looseTransgenic fish are farm-raised, isolated from wild stocksBut even during farming of wildtype fish, escapes happenfrequently (~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
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In experiments, transgenic males mated 3x morefrequently than the smaller wild malesOffspring of transgenic males lived <70% as long aswild malesCould lead to a decline of the wild fish population &endanger a species as whole
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http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html
+Antifreeze
wildtransgenic
Antifreeze Proteins (AFP)AFPs lower the freezing temperature of blood & fluidsTrout normally do not survive in water below –0.6°CTransgenic trout containing an AFP gene & promoter cansurvive in waters as cold as –1.2°C
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Conservation CloningMany endangered or extinct animals are being cloned orconsidered for cloningGaurBucardo mountain goat
MammothQuaggaBanteng
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Animal Bioreactors“Pharming”
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http://nolswf.bbc.net.uk/science/genes/gene_safari/pharm/a_pharming.shtml
1997, Tracy the sheep, the first transgenic animal toproduce a recombinant protein drug in her milkalpha-1-antitrypsin (AAT) treatment for emphysema &cystic fibrosisCreated by PPL Therapeutics & The Roslin Institute
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Webster and Peter
Nexia Biotechnologies transfered the silk gene from Orbspiders into goatsThe resulting male goats were used to sire silk-producingfemale goatsEach goat produces several grams of silk protein in hermilkThe silk is extracted, dried to a white powder, and spuninto fibersThe fibers are stronger and more flexible than steel
Transgenic male kidscarrying silk gene
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GTC Biotherapeutics has received approval to sell humananti-thrombin (ATryn) purified from goat’s milk in EuropeTechnology is not restricted to cows, goats, & sheepThere is interest in using rabbits since housing costs aresignificantly less & generation time is fasterChickens which produce recombinant drugs in their eggshave been produced by The Roslin Institute
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Other Types of Transgenic Animals
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Transgene ->Gene codingfor a growthhormone
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ANDi, the first transgenic primate born in January, 2000224 unfertilized rhesus eggs were infected with a GFP virus~Half of the fertilized eggs grew and divided40 were implanted into twenty surrogate mothersfive males were born,two were stillbornANDi was the only live monkey carrying the GFP gene
http://www.ohsu.edu/unparchive/2001/011001andi.shtml
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Alba, the EGFP (enhanced GFP) bunnyCreated in 2000 as a transgenic artwork
http://www.ekac.org/gfpbunny.html#gfpbunnyanchor
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http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Pigs Pass on the Transgene
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GloFish, originally developed in Singapore as a way to monitorwater pollutionThe normally black-and-silver zebrafish was turned green orred by inserting various versions of the GFP geneGlofish are on sale throughout the US except in CaliforniaGlofish retail for about $5 per fish. Normal zebrafish costaround one tenth of the price
http://www.nus.edu.sg/corporate/research/gallery/research12.htm
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Mouse “Knock-out” TechnologyGene Targeting
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Knock-out technology allows for the specific loss of a genein miceAllows for the function of the KO’d gene to be deduced fromthe defects seen in the micecan be used to mimick some diseaseUnlike traditional transgenics the trangene is targeted to aspecific site in the DNA of the mouse
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http://cba.musc.edu/SC_COBRE/CORE-B/Resources-B.htm
Mouse Knock-outs require embryonic stem (ES) cellsThese are derived from the inner cell mass (ICM) of ablastocyst (the ICM is what will become the fetus)ES cells are pluripotent meaning they can become all thedifferent cell types found in an adult
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http://tasq.uq.edu.au/blasto.html
Blastocyst Injection
Blastocyst ES cells
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http://bunseiserver.pharm.hokudai.ac.jp/gihou/knockout.html
Chimeric mouseThe brown fur comes from ES cells injectedinto the blastocyst of an albino mouse
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Some Examples of Knockout Mice
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p27 knockout mouse is bigger than the controlThis is not due to obesity, but the skeletal structure isincreased in size (everything about the mouse is larger)
http://www.bioreg.kyushu-u.ac.jp/saibouE.html
p27 knockout mouse
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http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
GDF8 (Myostatin) knockout mouseOver twice the muscle mass of a wildtype mouse
normal knockout
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Naturally Occurring GDF8 Mutants
http://www.canada.com/victoriatimescolonist/story.html?id=67f15c17-2717-4022-bb76-1b982456e793&k=94653http://www.bbc.co.uk/science/genes/gene_safari/wild_west/bigger_and_better02.shtml
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FGF5 knockout mouse has long, angora-like hair
http://www.med.uni-jena.de/ivm/deutsch/method/method_7.htm
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Clones and Cloning
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http://www.harlemlive.org/community/health-science/scientificcommunity/index2.html
Dolly as a lamb withher surrogate mother
Dolly, First Mammal Cloned From an Adult Cell
Dolly, as an adult
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http://www.howstuffworks.com/cloning3.htm
Somatic CellNuclear Transfer
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What Has Been Cloned So Far?
Somatic Cell Nuclear TransferSheep, Goat, Mouse, Rabbit, Cattle (domestic & wild),
Pig, Horse, Mule, Dog, Cat (domestic & wild), Deer
Embryo Splitting (Twinning)Sheep, Cattle, Primate (Rhesus)
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Cat Clone
Donor Surrogate mother with clone (CC)
Out of 87 implants only CC survived to birth
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http://www.usatoday.com/news/science/2003-01-21-cloned-cats_x.htm
Donor & Clone
Rainbow & CC
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Cloned transgenic cat containing red fluorescent protein
http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001
Transgenic Clones
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http://news.nationalgeographic.com/news/2003/05/0529_030529_muleclone.html
Idaho Gem, first cloned mule
1st try 134 implants 2 pregnancies, both failed2nd try 113 implantations 14 pregnancies, one birth
Surrogatemother(horse)
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In addition to cloning pets or prized livestock,researchers are looking to clone transgenic animalsThis would allow for more uniform expression oftransgenic genesNot all transgenic animals express their transgenicgenes at equal levelsAlso allows for the rapid expansion to large flocksor herds of transgenic animals
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http://www.newrivervalley.com/biotech/day1.html
Piglets clones created by PPL Therapeutics in 2000The piglets carry a silenced copy of alpha 1,3 galactosyltransferase, or GT, an enzyme involved in organ rejectionIn order to guarantee compatibility a second GT gene mustalso be silenced
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Mammoth QuaggaBucardo Gaur
http://www.personal.psu.edu/faculty/t/r/trp2/mammoth.jpeghttp://www.serragaucha.com.br/rocky/zoo.htmlhttp://www.riosmith.net/Gaur004.jpghttp://www1.ceit.es/Asignaturas/Ecologia/EspNaturales/Ordesa/mamiferos.htm#Bucardo
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http://www.advancedcell.com/images/Banteng002-sm.jpg
Noah, a Banteng clone created by Advanced Cell TechnologiesBanteng are endangered wild bovine from Southeast AsianThis clone was created from frozen tissue of an animal thatdied in 1980
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3 Pig clones, born in 2002, died of heart attacks due to “adultclone sudden death syndrome” within days of each other bythe time they were 6 months old.Dolly had a weight problem, telomeres 20% shorter thannormal, she suffered from arthritis, and finally lung cancerdue to an infection for which she was finally euthanized atage 6yrs.The success rate ranges from 1 to 3% this contrasts to invitro fertilization which has a success rate of 50 to 20%
Problems with Cloning
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http://fig.cox.miami.edu/~cmallery/150/gene/cloning.success.jpg
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Nearly all clones show some genetic anomaliesSome suffer from placental defects others cardiac defectsMany suffer from large offspring syndrome (LOS)
Normal mouse pup Cloned mouse pupsuffering from LOS