HC70A & SAS70A Winter 2009 Genetic Engineering in Medicine, Agriculture, and Law Professors Bob Goldberg & John Harada Lecture 8 Twenty-First Century Genetic Engineering Applications-Part Two Course AdministratorpTHEMES 1. Review Bacterial Genetic Engineering Applications 2. Genetic Engineering Applications Using Yeast a. What Are Fungi? Advantages or Genetic Engineering Human Genes & Proteins? b. Drugs & Vaccines c. Fermentation & Alcoholic Beverages d. Advantages 3. Genetic Engineering Applications Using Animals 4. Genetic Engineering Applications Using Plants 5. Genetic Engineering Humans-Gene Therapy 6. Gene Therapy With Small RNAs to Treat Human DiseasesSome Uses of Bacterial Genetic Engineering Review Of Genetic Engineering Applications Part One 1. New Drugs Using Human Proteins (e.g., insulin) 2. New Vaccines (e.g., cold virus) 3. New Antibiotics and Discovery of New Antibiotic-Producing Microbes (Metagenomics) 4. Food Products (Chymosin in Cheese) 5. Engineering Bacterial Metabolic Pathways For Industrial Applications (e.g., Dyes, Ethanol Production, Environmental Remediation) 6. Using Bacteria As a Delivery System For Counteracting Human Diseases (e.g., Inflammatory Bowel Bisease) and Tooth DecayFungi, or Molds are Classified as: a. Eukaryotes b. Prokaryotes c. Both Eukaryotes and ProkaryotesGenetic Engineering Yeast (Saccharomyces cerevisiae) Nucleus & NucleolusUsing Yeasts as Factories and “Catalysts” What Are Yeasts? Eukaryote 12 Mb Genome 6,000 Genes YeastFungi Are Human, Animal, and Plant Pathogens ~ 7,000 Human Deaths Annually in US Due To Fungal Infections 1. Candida albicans (bakers yeast relative) Infects AIDS Patients & Also Causes Vaginal Infections. Normally Present on Body (e.g., skin, mouth)..Anitbiotics and Steroids can “Induce” by Changing Bacteria That Keep Fungi Under Control 2. Cryptococcus, Aspergillus, & Histoplasma Next Most Common Fungi That Infect patients With Impaired Immune Systems 3. Coccidiodes (soil fungus)Can Cause Flu-like Symptoms 4. Antibiotics Do Not Work on Fungi (Why?) 5. New Anti-Fungal Drugs NeededFungi Can Devastate Crop Production-Major Cause of Reduced Yields Fungal Infected Soybeans Classical and Molecular Genetic Engineering Can Help Prevent Crop Loss Due To Fungi (How?)Yeast Can Be Used To Engineer Recombinant Drugs What Are the Advantages Over Bacteria? Vectors? Switches?Yeast Is Engineered Using YAC Vectors That Replicate in E. coli and Yeast YAC Vector = Yeast Artificial Chromosome That Can Accommodate ~ 100 kb of DNA and Behaves Like a Chromosome at Cell Divisions What Are the General Properties Of a Yeast Vector?Engineering Yeast Metabolic Pathways Could Be Beneficial to Humans Remember: Enzymes Encoded by Genes in Genome ∴ Genes Control Metabolic Processes Indirectly One Gene=One Enzyme Energy ProductionEthanol Energy All Energy is Derived Ultimately From the Sun7.2 Energy-Producing Metabolic Pathways Metabolic Pathways Leading to Energy Production Are in the Cytoplasm Energy Production Pathway is in the Mitochondria-Using Enzymes Encoded by Mitochondrial DNAYeast Fermentation in the Absence of Oxygen Plays a Major Role in Food and Beverage Productions ATP ⇒ Energy! Aerobic Anaerobic Bread Rising Champagnes Vinegar (Other Microbes) Alcoholic BeveragesYeast Plays a Major Role in Alcohol ProductionUsing Yeast to Make Alcoholic Beverages Cannot Grow Above 18% Ethanol? Some Can Grow to 25% (Fuel Production) Ethanol CH3CH2OH SugarsGenetic Engineering Yeast To Survive Above 18-25% Alcohol Why Isnʼt There GMO Wine & Beer With Higher/Lower Alcohol Content and/or New Flavors?Genetic Engineering of AnimalsUsing the Mouse as a Model to Determine the Function of Human GenesRecall-Human and Mouse Genomes 99% Similar ∴ Can Study Human Genes Using Mouse as a ModelHow Many Human Disease Genes Have Been Identified? There are ~25,000 Genes in The Human Genome 1. ~2,700 Genes Correlate With a Disease Phenotype 2. The Molecular Basis of 90% of These Genetic Diseases Are Known (e.g., Sickle Cell Anemia, Hemophilia A) Nature Education!1(1),!(2008) http://www.nature.com/scitableThe Human Genome Contains ~25,000 Different Genes Do Not Know Functions of Most Human Genes!Genetic Engineering Mice and Other Mammals 1. Egg DNA Injection 2. Embryo Stem Cell DNA Transformation Both Methods Alter the Germ Line (i.e., Genes are Inherited)Transgenic Mice and Other Mammals Need To Be Tested For the Presence of the Transgene DNA Fingerprint Test Using PCRMouse Genes Related to Those in the Human Genome Can be “Knocked Out”In Order to Determine Their Functions Insertional Mutagenesis (Similar to Mutating the β-gal gene Producing White Colonies) Gene Related to a Human Disease Gene Mutant Gene Transfer to Mouse & Observe PhenotypeAnimals Can Also be Used as Factories to Produce Large Amounts of Human Proteins 1. Many human proteins need to be modified after translation to be active. Only eukaryotic cells can do this. 2. Bacteria need big fermentors + elaborate protein purification schemes--Farm animals can be used for this purpose w/o special processing/machinery. 3. Proteins stable, can be made in large amounts, and purified easily Advantages of Molecular Pharming Where is Switch Active?Genetically Engineered Drug-Producing Mammals Can Also Be ClonedIf Cloning Humans Was 100% Safe and “Normal” Humans Could be Produced at the Same Percentage as Doing It “Naturally,” I Would Not Object To Individuals Cloning Themselves, Parents, Children, or Whomever They Wanted to Clone: a. Yes b. NoMaking Recombinant Human Proteins in Animals Advantages over Bacteria? !!!Making Transgenic Mammals is an Inefficient Process Hypothesis For Inefficiency?Examined Data From Seven Generations of Genetically Engineered Goats Issues Food Supply? Containment? Anima Health? Effective Drug?Blood Clotting and Anti-Thrombin? Eight Proteins/Genes Required: 1. Factor VII 2. Factor XI 3. Factor IX 4. Factor VIII 5. Factor X 6. Protein C 7. Prothrombin 8. Fibrinogen Anti-Thrombin Deficiency (At-III) Genetic Disease Atryn® Goat Drug Post-translational Modification Inactive Active Hemophilia A Hemophilia B Thrombin Must Be Kept Inactive In Absence of Wound XOther Transgenic Animals Have Been GeneratedGenetic Engineering Transgenic Salmon What Are the Issues With These Fish? Engineered With Growth Hormone Gene Superfish