HC70A

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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 Administratorp
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THEMES

• Review Bacterial Genetic Engineering Applications
  
• Genetic Engineering Applications Using Yeast
• What Are Fungi? Advantages or Genetic
  Engineering Human Genes Proteins?
• Drugs Vaccines
• Fermentation Alcoholic Beverages
• Advantages
• Genetic Engineering Applications Using Animals
• Genetic Engineering Applications Using Plants
• Genetic Engineering Humans-Gene Therapy
• Gene Therapy With Small RNAs to Treat Human
  Diseases

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Some Uses of Bacterial Genetic EngineeringReview
Of Genetic Engineering ApplicationsPart One

• New Drugs Using Human Proteins (e.g., insulin)
• New Vaccines (e.g., cold virus)
• New Antibiotics and Discovery of New
  Antibiotic-Producing Microbes (Metagenomics)
• Food Products (Chymosin in Cheese)
• Engineering Bacterial Metabolic Pathways For
  Industrial Applications (e.g., Dyes, Ethanol
  Production, Environmental Remediation)
• Using Bacteria As a Delivery System For
  Counteracting Human Diseases (e.g., Inflammatory
  Bowel Bisease) and Tooth Decay

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• Fungi, or Molds are Classified as
• a. Eukaryotes
• Prokaryotes
• Both Eukaryotes and Prokaryotes

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Genetic Engineering Yeast (Saccharomyces
cerevisiae)
Nucleus Nucleolus
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Using Yeasts as Factories and CatalystsWhat
Are Yeasts?
Yeast
Eukaryote 12 Mb Genome 6,000 Genes
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Fungi Are Human, Animal, and Plant Pathogens

• 7,000 Human Deaths Annually in US Due To Fungal
  Infections
• 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
• Cryptococcus, Aspergillus, Histoplasma Next
  Most Common Fungi That Infect patients With
  Impaired Immune Systems
• Coccidiodes (soil fungus)Can Cause Flu-like
  Symptoms
• Antibiotics Do Not Work on Fungi (Why?)
• New Anti-Fungal Drugs Needed

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Fungi Can Devastate Crop Production-Major Cause
of Reduced Yields
Classical and Molecular Genetic Engineering Can
Help Prevent Crop Loss Due To Fungi (How?)
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Yeast Can Be Used To Engineer Recombinant Drugs
What Are the Advantages Over Bacteria?
Vectors? Switches?
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Yeast Is Engineered Using YAC Vectors That
Replicate in E. coli and Yeast
What Are the General Properties Of a Yeast Vector?
YAC Vector Yeast Artificial Chromosome That Can
Accommodate 100 kb of DNA and Behaves Like a
Chromosome at Cell Divisions
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Engineering Yeast Metabolic Pathways Could Be
Beneficial to Humans
RememberEnzymes Encoded by Genes in Genome ?
Genes Control Metabolic Processes Indirectly One
GeneOne Enzyme
Energy Production
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All Energy is Derived Ultimately From the Sun
Ethanol
Energy
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7.2 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 DNA
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Yeast Fermentation in the Absence of Oxygen Plays
a Major Role in Food and Beverage Productions
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Yeast Plays a Major Role in Alcohol Production
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Using Yeast to Make Alcoholic Beverages
Sugars
Ethanol
Cannot Grow Above 18 Ethanol? Some Can Grow to
25 (Fuel Production)
CH3CH2OH
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Genetic Engineering Yeast To Survive Above 18-25
Alcohol
Why Isnt There GMO Wine Beer With Higher/Lower
Alcohol Content and/or New Flavors?
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Genetic Engineering of Animals
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Using the Mouse as a Model to Determine the
Function of Human Genes
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Recall-Human and Mouse Genomes 99 Similar ? Can
Study Human Genes Using Mouse as a Model
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How Many Human Disease Genes Have Been Identified?

• There are 25,000 Genes in The Human Genome
• 2,700 Genes Correlate With a Disease Phenotype
• 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/scitable
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The Human Genome Contains 25,000 Different Genes
Do Not Know Functions of Most Human Genes!
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Genetic Engineering Mice and Other Mammals
Both Methods Alter the Germ Line (i.e., Genes are
Inherited)
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Transgenic Mice and Other Mammals Need To Be
Tested For the Presence of the Transgene
DNA Fingerprint Test Using PCR
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Mouse Genes Related to Those in the Human Genome
Can be Knocked Out In Order to Determine Their
Functions
Gene Related to a Human Disease Gene
Insertional Mutagenesis (Similar to Mutating the
?-gal gene Producing White Colonies)
Transfer to Mouse Observe Phenotype
Mutant Gene
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Animals Can Also be Used as Factories to Produce
Large Amounts of Human Proteins
Advantages of Molecular Pharming
Where is Switch Active?
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
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Genetically Engineered Drug-Producing Mammals
Can Also Be Cloned
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• If 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
• Yes
• No

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Making Recombinant Human Proteins in Animals
Advantages over Bacteria?
!!!
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Making Transgenic Mammals is an Inefficient
Process
Hypothesis For Inefficiency?
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Issues Food Supply? Containment? Anima
Health? Effective Drug?
Examined Data From Seven Generations of
Genetically Engineered Goats
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Blood Clotting and Anti-Thrombin?

• Eight Proteins/Genes Required
• Factor VII
• Factor XI
• Factor IX
• Factor VIII
• Factor X
• Protein C
• Prothrombin
• Fibrinogen

Post-translational Modification
Inactive
Active
Hemophilia A
Hemophilia B
X
Thrombin Must Be Kept Inactive In Absence of Wound
Anti-Thrombin Deficiency (At-III) Genetic Disease
Atryn? Goat Drug
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Other Transgenic Animals Have Been Generated
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Genetic Engineering Transgenic Salmon
Engineered With Growth Hormone Gene
What Are the Issues With These Fish?
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Myths About Transgenic Salmon
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How Genes Work What Are Genes In Context of
Thinking About The Consequences of GMOs

• What is a Gene?
• What is the Anatomy of a gene?
• How Does the Gene Replicate?
• How Does the Gene Direct Synthesis of a Protein?
• Does the Gene Work Independently of other Genes?
• What is the Sequence Structure of the Protein?
• How does it work in cell?
• Does the Protein Structure imply any Potential
  Harm?
• Does the Gene Change the organism? Fitness?

Need Science- Based Questions Science-Based
Solutions-NOT OPINIONS!
Behind All Traits!
Theres NO HOCUS POCUS all hypothesis are
testable!!
Same Processes!
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Genetic Engineering of Plants
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Plants Have Been Engineered For Large Numbers of
Traits in Laboratories Around the World Tens of
Thousands of GE Experiments!!
(Canola)
Decaffeinated Coffee
Allergen-free seeds/grains
Drought Resistance
Ethanol/Transportation Fuel
But Only a Few Have Helped Generate New
Crops! The Simple Ones With Economic Drivers
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One Way is to Use These New Traits in Engineered
Crops That Farmers Have Adopted Faster Than Any
New Agricultural Technology In the Past 100 Years!
Over 2 Billion Acres of Bioengineered Crops Have
Been Grown World-Wide Since 1996 and 300 Million
Acres in 2008
ISAAA Brief, 2007
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Engineered Crops Have Increased Yields, Reduced
Pesticide Use, and Increased Incomes of Farmers
in the Developing World
United Nations FAO Report No. 35, 2003-04
Scientific American, September, 2007
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Specific Examples of Bioengineered CropsPest
Resistance
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How to Make an Insect-Resistant Plant
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Engineering Papaya For Resistance to Papaya
Ringspot Virus
Saved Hawaiian Papaya Industry
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Using a Wild Potato Gene to Engineer Potato
Plants Resistant to Potato Blight Fungus
Resistant Transgenic Plant
Sensitive Non-Transgenic Control
Potato Blight Caused the Irish Famine That Killed
One Million People in the Late 19th Century and
Resulted in a Large Migration of Irish People to
the United States!!!
Gene RB Cloned From Solanum bulbocastanum
Confers Broad Spectrum Resistance to Potato Late
Blight Song et al., PNAS 100, 9128-9133 (2003)
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Specific Examples of Bioengineered CropsAbiotic
Stress
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Identifying Genes For Drought and Freezing
Tolerance Major Factors in Lowering Crop Yield
Zhang et al. Plant Physiology 135, 615-621 (2004)
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Identifying Salt Tolerant Genes

Sanan-Mishra et al. PNAS 102, 509-514 (2005)
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Specific Examples of Bioengineered Crops Seeds
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Vitamin A Deficiency Causes 1,000,000 Deaths Per
Year!
Genetic Engineering a Seed Metabolic Pathway!
Other Nutritionally-Enhanced Seeds (e.g.,
Folates, Micronutrients, Vitamin E)
Kinney AJ..Curr Opin Biotechnol. (2006) 17,
130-8.
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Specific Examples of Bioengineered Crops
Biofuels
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The Perfect Energy Crop
High biomass increased growth rate,
photosynthetic efficiency, delayed flowering
Rapid and cost-effective propagation
Improved composition structure higher fuel
yield per ton
Stand establishment cold germination, cold growth
Disease and pest resistance
Perennial multi-year crop, efficient nutrient
use, high fossil energy ratio
Optimized architecture dense planting, no
lodging, easier harvest
Deep roots drought tolerance, nutrient uptake,
carbon sequestration
Salt, pH and Aluminum tolerance
These Are All Yield Traits! Identify From Genome
Projects!
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Engineering Biomass 2009 Back to the Future!
Engineering Biomass 10,000 Years Ago
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Re-Engineering Plants as Drug Factories
Back to the Future!!
Aspirin? Willow Bark!
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Plants Can Also be Used as Factories to Produce
Large Amounts of Human Proteins Similar to Goats
Molecular Pharming
Where is Switch Active?
Switch?
Reasons
Advantages
1. 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
plants can be used for this purpose w/o special
processing/ machinery 3. Proteins in plants (e.g.
seeds) infinitely stable- can be stored cheaply
(grown cheaply) for long periods of time!
Modifications
Cost
Stability
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Pharming in Plants
Advantages
1. Cost 2. Simplicity of Method 3. Stability of
Proteins
Vaccines
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Advantages For Developing World Combating
Biowarfare?
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Other Human and Animal Vaccines Are Being
Engineered in Plants
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HoweverTheres a Battle Raging to Get
Bioengineered Crops Adopted in Many Parts of the
World
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Safety Issues of Genetically Engineered Plants
Have Been Investigated and Discussed For Almost
25 Years!!!
1982
1986
1984
1985
2002
2001
2004
1987
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The GMO Controversy Has European Origins and is
Complex and Not Science Based

• Ideology / Anti-Technology / Anti-Biotech/Anti-Sci
  ence/Unnatural
• Lack of Confidence in Government -- No Strong
  USDA, FDA, or EPA Tradition in Europe (Protect
  Food Supply -- Mad Cow -- Dioxin)!
• Mistrust of International / US Corporations /
  Anti-Market -- Taking Over the Food Supply --
  Anti-Globalization -- Anti-Patent/Intellectual
  Property
• Labeling -- Want to Know and Choose What is Eaten
  (Personal Liberty)!
• Experience of Europe in WWII -- Wary of Genetic
  Manipulation
• Small Farmer Tradition in Europe
• Production-Oriented Farming -- Subsidies/More
  Production/More Euros (54B/2003)
• Organic Growers/Markets -- Gain Market Share
  (Follow the !!)/Pollen Flow --Contamination
• No Obvious Consumer Benefit -- First
  Generation AgBiotec/No Need

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What Has Been Some of the Real Life Affects of
the GMO Controversy?
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Professor Frank Furedi, University of Kent,
England
Professor Frank Furedi, University of Kent,
England
Professor Frank Furedi, University Of Kent,
England
Professor Frank Furedi, University Of Kent,
England
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Human Genetic Engineering
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? ?
Issues Regulation? NIH Guidelines?Human
Experimentation? Ethics?Eugenics?
a. b. c. d.
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In Vivo Cystic Fibrosis Gene Therapy
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In Vivo Cancer Suicide Cell Gene Therapy
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Suicide Gene Therapy
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Ex Vivo vs. In Vivo Somatic Cell Gene Therapy
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Ex Vivo vs. In Vivo Somatic Cell Gene Therapy
In vivo
Ex vivo
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Ex Vivo Gene Therapy for Severe Combined
Immunodeficiency (SCID)
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Humans Have Been Genetically Engineered To Cure a
Lethal Genetic Disease (SCID)
Several Teenagers Are Alive Because They Have
Been Engineered With an ADA Gene That They Were
Not Born With!!!
The Age of Human Genetic Engineering Began
Almost Twenty Years Ago Treating Severe Combined
Immunodeficiency Disease (SCID) With Normal ADA
Genes!!!
Adenosine Deaminase Gene (ADA)
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Retroviruses
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Retrovirus Life Cycle
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HIV is a Retrovirus
T-Cell
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Using Retroviruses for Ex Vivo Gene Therapy

• Cloning in Bacteria
• DNA Transformation into Packaging Cell

A.

• Packaging Cells Makes Viral Proteins
• Cannot Package (?-Minus)
• Packages Therapeutic Transcript (?-Plus)

B.
C.

• Infect Target Cells
• Check For Presence of Gene
• Transfer To Patient

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Types of Human Gene Therapy Clinical Trials
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Approved Gene Therapy Trials
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Approved Gene Therapy Trials By Disease and Vector
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Combining Gene Therapy With Stem Cells
Therapeutic Cloning
Genetic Engineer Cells Before Nuclear or Cell
Transfer
Example Defective Insulin Gene in Pancreas
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Human Gene Therapy Issues

• Regulation
• Consent
• Risks
• Enhancement
• Eugenics

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A Molecular Drug To Shut Off Genes-RNAi (e.g.,
Disease Genes, Viral Genes)
Lou Gehrigs Disease Amyotophic Lateral
Sclerosis (ALS) Caused by Dominant Mutations In
the Coding Region of the Superoxide Dismutase
(SOD1) Gene SOD1 Protein is Toxic To Motor
Neurons
If Mutant Gene Could Be Shut Off With a
Molecular Drug, Disease Might Not Develop
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Using Antisense RNA or RNAi To Inhibit Gene
Activity
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Using RNAi To Inhibit Gene Activity
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