Transgenic Organisms

Definitions: Genetically modified organisms (GMO’s): -Organisms whose genes have been altered using genetic engineering techniques. Transgenic organisms - Most GMO’s are transgenic organisms… they have received genes from a different organism. Cry genes (produce Bt toxin) from Bacillus thurengensis (Bt) put into peanut plant - European corn borer larvae Genetically engineered sheep with the human gene responsible for the production of alpha-1-antitrypsin (AAT) transferred into their DNA. When the AAT is eventually extracted from their milk, it will be used as therapy for humans deficient in AAT. The deficiency causes emphysema (a breathing disorder) in approximately 100,000 people in the western world. Ex. A mouse is given a gene from a human. The mouse is a transgenic GMO. Trans- ; across (across species in this case)

How Do They Do It?

GMO’s at home: GloFish Zebra danio 1. Zebra danio was genetically engineered with a gene from sea coral that causes the fish to glow in the presence of environmental toxins. 2. Gene was inserted into the embryo of the fish. 3. First GMO available as a pet.

(jellyfish, phylum cnidaria) GMO’s in research: GFP (green fluorescent protein) GFP Mice 1. Gene from a jellyfish (Aequorea victoria) that codes for GFP was inserted into the embryos of mice. Aequorea victoria (jellyfish, phylum cnidaria)

GMO’s in research: 1. GFP is used in cellular and molecular biology. GFP (green fluorescent protein) – a reporter protein 1. GFP is used in cellular and molecular biology. 2. You can attach this protein to any other protein you want making it a reporter protein. - It “reports” to you where the protein is going since it emits green light (similar to radioactivity in that sense)

GMO’s in research: - GFP has been attached to a protein called MFD, which is found in peroxisomes. - Those little green dots are peroxisomes… - You can track any protein you want…in a single cell or an entire organism

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                                                         

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

As water temperature drops the GH gene is turned on 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 wild type 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 http://pubs.acs.org/hotartcl/chemtech/99/jun/fletcher.html

This is a genetically modified strain of malaria-resistant mosquito which has been created successfully by a scientist. The creation of mosquitoes with green fluorescent testicles will help curb the spread of malaria-carrying mosquitoes.

Animal Bioreactors “Pharming”

GMO medicine: E. Coli with the human insulin gene Cry genes (produce Bt toxin) from Bacillus thurengensis (Bt) put into peanut plant - European corn borer larvae Genetically engineered sheep with the human gene responsible for the production of alpha-1-antitrypsin (AAT) transferred into their DNA. When the AAT is eventually extracted from their milk, it will be used as therapy for humans deficient in AAT. The deficiency causes emphysema (a breathing disorder) in approximately 100,000 people in the western world. Alpha 1-Antitrypsin or α1-antitrypsin (A1AT) is a glycoprotein and generally known as serum trypsin inhibitor. Alpha 1- antitrypsin is also referred to as alpha-1 proteinase inhibitor (A1PI) because it is a serine protease inhibitor (serpin), inhibiting a wide variety of proteases.[1] It protects tissues from enzymes of inflammatory cells, especially elastase, and has a reference range in blood of 1.5 - 3.5 gram/liter (in US the reference range is generally expressed as mg/dL or micromoles), but the concentration can rise manyfold upon acute inflammation.[2] In its absence, elastase is free to break down elastin, which contributes to the elasticity of the lungs, resulting in respiratory complications such as emphysema, or COPD (chronic obstructive pulmonary disease) in adults and cirrhosis in adults or children. - Insulin is made using the bacterium E. coli. - The human gene coding for insulin is inserted into E. coli, which will then make insulin for us (we will see how this is done shortly)…

GMO medicine: AAT Sheep Cry genes (produce Bt toxin) from Bacillus thurengensis (Bt) put into peanut plant - European corn borer larvae Genetically engineered sheep with the human gene responsible for the production of alpha-1-antitrypsin (AAT) transferred into their DNA. When the AAT is eventually extracted from their milk, it will be used as therapy for humans deficient in AAT. The deficiency causes emphysema (a breathing disorder) in approximately 100,000 people in the western world. Alpha 1-Antitrypsin or α1-antitrypsin (A1AT) is a glycoprotein and generally known as serum trypsin inhibitor. Alpha 1- antitrypsin is also referred to as alpha-1 proteinase inhibitor (A1PI) because it is a serine protease inhibitor (serpin), inhibiting a wide variety of proteases.[1] It protects tissues from enzymes of inflammatory cells, especially elastase, and has a reference range in blood of 1.5 - 3.5 gram/liter (in US the reference range is generally expressed as mg/dL or micromoles), but the concentration can rise manyfold upon acute inflammation.[2] In its absence, elastase is free to break down elastin, which contributes to the elasticity of the lungs, resulting in respiratory complications such as emphysema, or COPD (chronic obstructive pulmonary disease) in adults and cirrhosis in adults or children. Genetically engineered sheep with the human gene for alpha-1-antitrypsin (AAT). AAT is extracted from their milk and used to treat humans deficient in AAT, which is one cause of emphysema (a breathing disorder) in approximately 100,000 people in the western world.

GMO’s and medicine: Transgenic cows can make serum albumin for treatment of burns and severe trauma to tissue

Other Types of Transgenic Animals

Spider silk Spider’s silk is 5 times stronger than a thread of steel of the same thickness 3 times stronger than Kevlar (carbon fibre) Spiders are carnivores and cannot be raised Transgenic animals can produce the spider protein Aculepeira ceropegia a European orbweb spider © P. Billiet © 2010 Paul Billiet ODWS

Spider silk The gene for spider silk protein was isolated from the golden orb weaver (Nephila clavipes) It is a fibrous protein so it has a very repetitive gene sequence Nephila clavipes © C. Frank Starmer © 2010 Paul Billiet ODWS

Web-based kid Spider goat © Sean O'Neill Gene (plus regulator genes) inserted into goat mammary gland cells Genetically transformed cells could be made to secrete spider silk protein Transformed goat cells fused to enucleated oocyte Genetically transformed goat embryos produced © 2010 Paul Billiet ODWS

Applications Super strong surgery thread Artificial ligaments Light bullet proof body armor Biodegradable fishing line © 2010 Paul Billiet ODWS

ANDi, the first transgenic primate born in Jan 2000                                                  ANDi, the first transgenic primate born in Jan 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

Researchers transfer DNA from the long-vanished Tasmanian tiger into a mouse. The finding shows how lost information about species from the past can be retrieved and also provides a glimpse into how long-gone creatures may someday get a second chance at life.

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 mimic 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

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 Organisms

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

GDF8 (Myostatin) knockout mouse normal knockout GDF8 (Myostatin) knockout mouse Over twice the muscle mass of a wild type 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

Conclusion - We can basically move any gene(s) between members of a species or between any species. - We can also alter the genes to our liking (GFP tagged proteins) before inserting them into embryos. Cry genes (produce Bt toxin) from Bacillus thurengensis (Bt) put into peanut plant - European corn borer larvae Genetically engineered sheep with the human gene responsible for the production of alpha-1-antitrypsin (AAT) transferred into their DNA. When the AAT is eventually extracted from their milk, it will be used as therapy for humans deficient in AAT. The deficiency causes emphysema (a breathing disorder) in approximately 100,000 people in the western world. Is all of this genetic engineering positive or negative?