What About the Term Genetic Engineering? Genetic engineering is the basic tool set of biotechnology Genetic engineering involves: Isolating genes Modifying genes so they function better Preparing genes to be inserted into a new species Developing transgenes Genetic engineering is the collection of techniques necessary to create a transgene. These procedures include isolating the gene-of-interest from the tens of thousands of genes found in the genome of a species. Once that gene is isolated, it is modified so it functions better in an organism. That gene is then mixed with other genes to prepare it to be introduced into another organism. This whole step develops transgenes.

What is a transgenic? Concept Based on the Term Transgene Transgene – the genetically engineered gene added to a species Ex. – modified EPSP synthase gene (encodes a protein that functions even when plant is treated with Roundup) Transgenic – an organism containing a transgene introduced by technological (not breeding) methods Here I am pointing out the difference between the process (using a transgene) and the product (the transgenic plant). Ex. – Roundup Ready Crops

Why are transgenics important? We can develop organisms that express a “novel” trait not normally found in the species Extended shelf-life tomato (Flavr-Savr) It is important to separate the process from the product. Focus on the product, it will highlight the benefits of biotechnology. Herbicide resistant soybean (Roundup Ready)

Environmental Cleanup Biotechnology is Not Just on the Farm Disease Treatment Diagnostics   Environmental Cleanup Human Applications A list of human and environmental applications of biotechnology.

Human Applications Pharmaceutical products New solutions to old problems   Disease diagnosis Determine what disease you have or may get  Gene therapy Correcting disease by introducing a corrective gene These are the general classes of human applications. Pharmaceutical products will be discussed on the next slide. Disease diagnosis using the tools of biotechnology to determine if an individual is at risk for contracting a certain disease. In some cases, the result will predict a 100% probability, whereas in other cases a positive result means a significantly increased risk. Gene therapy involves the insertion of a functioning gene into a human. The gene provides a protein product that is not being produced in the individual because of a mutation in their corresponding gene.

Biotechnology and Health Product Use Insulin Diabetes Interferon Cancer Interleukin Human growth hormone Dwarfism Neuroactive proteins Pain Each of these products were developed by the pharmaceutical industry using biotechnology approaches. Most of these are engineered into bacteria, and the protein product is extract in large quantities for future sale. The genes for these proteins are: Cloned Inserted into bacteria Product isolated using biofermentation

Environmental Applications Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution Indicator bacteria – contamination can be detected in the environment Recombinant (genetically-engineered) bacteria have been developed that help clean up contaminated sites. They can also be used to determine if a specific site is contaminated with a specific pollutant.

Future Health-related Biotech Products Vaccines – herpes, hepatitis C, AIDS, malaria Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel The health and pharmaceutical industry uses biotech approaches for vaccine production. The normal bacteria in the mouth produces an acid that destroys enamel. The engineered version of the bacteria does not produce the acid. Children will be treated with the engineered version, which also has a gene that will destroy the other bacteria in the mouth, allowing it to become established.

Transgenic Plants Serving Human Health Needs Edible Vaccines Transgenic Plants Serving Human Health Needs Works like any vaccine A transgenic plant with a pathogen protein gene is developed Potato, banana, and tomato are targets Humans eat the plant The body produces antibodies against pathogen protein Humans are “immunized” against the pathogen Examples: Diarrhea Hepatitis B Measles Edible vaccines may be the most important and accepted biotech product. The principles are the same as those used for normal vaccines: a protein enters the body in some manner, and the human immune system produces antibodies against that protein. When the human is then exposed to the pathogen, the immune system is turned on and destroys the pathogen.

A Popular Term We Need To Know GMOs - Genetically modified organisms GMO - an organism that expresses traits that result from the introduction of foreign DNA Originally a term equivalent to transgenic organism GMO: notice that foreign DNA is a requirement for this definition. Plant breeding products are not GMOs, as some would like to claim.

Delivering the Gene to the Plant Transformation cassettes are developed in the lab They are then introduced into a plant Two major delivery methods Agrobacterium Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens. The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun. Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants. Tissue culture required to generate transgenic plants Gene Gun

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

PROTOPLAST preparation

Gene Gun

The Lab Steps And this slide illustrates those steps.

The Next Test Is The Field Herbicide Resistance Non-transgenics The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants. Transgenics

Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn What is needed is for the public to accept these crops. Examples such as these, were a corn crop is freed of weed pressure make a compelling case for acceptance of these new agricultural products. But, it should be noted that these traits are all producer orientated. Before After

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

Antifreeze Proteins 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”

alpha-1-antitrypsin (AAT) treatment for emphysema & cystic fibrosis 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

The resulting male goats were used to sire silk-producing female goats 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 Webster and Peter Transgenic male kids carrying silk gene

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, 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 are on sale throughout the US except in California 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

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

GDF8 (Myostatin) knockout mouse 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

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

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