Title: HC70A
1 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
2THEMES
- 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
3Some 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
4- Fungi, or Molds are Classified as
- a. Eukaryotes
- Prokaryotes
- Both Eukaryotes and Prokaryotes
5Genetic Engineering Yeast (Saccharomyces
cerevisiae)
Nucleus Nucleolus
6Using Yeasts as Factories and CatalystsWhat
Are Yeasts?
Yeast
Eukaryote 12 Mb Genome 6,000 Genes
7Fungi 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
8Fungi Can Devastate Crop Production-Major Cause
of Reduced Yields
Classical and Molecular Genetic Engineering Can
Help Prevent Crop Loss Due To Fungi (How?)
9Yeast Can Be Used To Engineer Recombinant Drugs
What Are the Advantages Over Bacteria?
Vectors? Switches?
10Yeast 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
11Engineering Yeast Metabolic Pathways Could Be
Beneficial to Humans
RememberEnzymes Encoded by Genes in Genome ?
Genes Control Metabolic Processes Indirectly One
GeneOne Enzyme
Energy Production
12All Energy is Derived Ultimately From the Sun
Ethanol
Energy
137.2 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
14Yeast Fermentation in the Absence of Oxygen Plays
a Major Role in Food and Beverage Productions
15Yeast Plays a Major Role in Alcohol Production
16Using Yeast to Make Alcoholic Beverages
Sugars
Ethanol
Cannot Grow Above 18 Ethanol? Some Can Grow to
25 (Fuel Production)
CH3CH2OH
17Genetic Engineering Yeast To Survive Above 18-25
Alcohol
Why Isnt There GMO Wine Beer With Higher/Lower
Alcohol Content and/or New Flavors?
18Genetic Engineering of Animals
19Using the Mouse as a Model to Determine the
Function of Human Genes
20Recall-Human and Mouse Genomes 99 Similar ? Can
Study Human Genes Using Mouse as a Model
21How 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
22The Human Genome Contains 25,000 Different Genes
Do Not Know Functions of Most Human Genes!
23Genetic Engineering Mice and Other Mammals
Both Methods Alter the Germ Line (i.e., Genes are
Inherited)
24Transgenic Mice and Other Mammals Need To Be
Tested For the Presence of the Transgene
DNA Fingerprint Test Using PCR
25Mouse 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
26Animals 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
27Genetically Engineered Drug-Producing Mammals
Can Also Be Cloned
28- 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
29Making Recombinant Human Proteins in Animals
Advantages over Bacteria?
!!!
30Making Transgenic Mammals is an Inefficient
Process
Hypothesis For Inefficiency?
31Issues Food Supply? Containment? Anima
Health? Effective Drug?
Examined Data From Seven Generations of
Genetically Engineered Goats
32Blood 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
33Other Transgenic Animals Have Been Generated
34Genetic Engineering Transgenic Salmon
Engineered With Growth Hormone Gene
What Are the Issues With These Fish?
35Myths About Transgenic Salmon
36How 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!
37Genetic Engineering of Plants
38Plants 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
39One 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
40Engineered 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
41Specific Examples of Bioengineered CropsPest
Resistance
42How to Make an Insect-Resistant Plant
43Engineering Papaya For Resistance to Papaya
Ringspot Virus
Saved Hawaiian Papaya Industry
44Using 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)
45Specific Examples of Bioengineered CropsAbiotic
Stress
46Identifying Genes For Drought and Freezing
Tolerance Major Factors in Lowering Crop Yield
Zhang et al. Plant Physiology 135, 615-621 (2004)
47Identifying Salt Tolerant Genes
Sanan-Mishra et al. PNAS 102, 509-514 (2005)
48Specific Examples of Bioengineered Crops Seeds
49Vitamin 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.
50Specific Examples of Bioengineered Crops
Biofuels
51The 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!
52Engineering Biomass 2009 Back to the Future!
Engineering Biomass 10,000 Years Ago
53Re-Engineering Plants as Drug Factories
Back to the Future!!
Aspirin? Willow Bark!
54Plants 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
55Pharming in Plants
Advantages
1. Cost 2. Simplicity of Method 3. Stability of
Proteins
Vaccines
56Advantages For Developing World Combating
Biowarfare?
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58Other Human and Animal Vaccines Are Being
Engineered in Plants
59HoweverTheres a Battle Raging to Get
Bioengineered Crops Adopted in Many Parts of the
World
60Safety Issues of Genetically Engineered Plants
Have Been Investigated and Discussed For Almost
25 Years!!!
1982
1986
1984
1985
2002
2001
2004
1987
61The 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
62What Has Been Some of the Real Life Affects of
the GMO Controversy?
63Professor 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
64Human Genetic Engineering
65? ?
Issues Regulation? NIH Guidelines?Human
Experimentation? Ethics?Eugenics?
a. b. c. d.
66In Vivo Cystic Fibrosis Gene Therapy
67In Vivo Cancer Suicide Cell Gene Therapy
68Suicide Gene Therapy
69Ex Vivo vs. In Vivo Somatic Cell Gene Therapy
70Ex Vivo vs. In Vivo Somatic Cell Gene Therapy
In vivo
Ex vivo
71Ex Vivo Gene Therapy for Severe Combined
Immunodeficiency (SCID)
72Humans 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)
73Retroviruses
74Retrovirus Life Cycle
75HIV is a Retrovirus
T-Cell
76Using 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
77Types of Human Gene Therapy Clinical Trials
78Approved Gene Therapy Trials
79Approved Gene Therapy Trials By Disease and Vector
80Combining Gene Therapy With Stem Cells
Therapeutic Cloning
Genetic Engineer Cells Before Nuclear or Cell
Transfer
Example Defective Insulin Gene in Pancreas
81Human Gene Therapy Issues
- Regulation
- Consent
- Risks
- Enhancement
- Eugenics
82A 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
83Using Antisense RNA or RNAi To Inhibit Gene
Activity
84Using RNAi To Inhibit Gene Activity
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