HC70A

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HC70A SAS70A Winter 2009 Genetic Engineering
in Medicine, Agriculture, and Law Professors
Bob Goldberg John Harada Lecture
7 Twenty-First Century Genetic Engineering
Applications-Part One Course Administratorp
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THEMES

• Review Origins of Genetic Engineering
• Overview of Genetic Engineering Applications and
  Uses Discussed to Date (e.g., DNA Fingerprinting,
  Screening For Disease Genes, Human Origins)
• Genetic Engineering Applications Using Bacteria
• Drugs, Vaccines, Antibiotics
• FDA Approval
• Antibody Production, Immune Responses, Gene
  Rearrangements, and Cancer
• Antibiotic Action Problems
• Metagenomics-Screening For New Bacteria
• Food Products (e.g., cheese)
• Metabolites, Biofuel Production, Bioremediation
• Human Drug Delivery Systems
• Genetic Engineering Applications Using Yeast
• What Are Fungi? Advantages or Genetic
  Engineering Human Genes Proteins?
• Drugs
• Fermentation Alcoholic Beverages

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Genetic Engineering Where Are We in Our
Discussion of Genetic Engineering and Its Uses?
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Why Clone Genes From An Organisms Genome? A
Review

• PURIFY Individual Genes From the Genome (
  e.g.,one of 25,000 human genes)
• AMPLIFY Genes to Obtain DNA (genes) For Study
• USE the Cloned Genes To
• Study Gene Structure Function ( THE Major Use!)
• Use to Sequence Genomes and Compare Genomes
• Use to Convert Cells Into Factories To Make Drugs
  and Pharmaceuticals
• Use to Screen For Genetic Diseases
• Use to Identify Individuals (e.g., Paternity,
  Forensics, Human Origins)
• Use to Correct Genetic Diseases
• Use to Engineer New Crops and Farm Animals
• Use to Identify and Sequence Ancient DNA
• And Many Other UsesAs You will See in the Next
  Two Lectures!!

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Genetic Engineering Has Affected Our Lives in
Many Ways-A Review!

• Basic Understanding of Living Processes and
  Ourselves
• Basic Understanding of Genes and Their Functions
• The Era of Genomics and the Sequence of the Human
  Genome and Those of Other Organisms
• Basic Understanding of Human Diseases Such as
  Cancer and Novel New Treatments
• A Multibillion Dollar Biotechnology Industry
• New Legal Issues Such as Genetic Privacy,
  Forensics, and Patents on Genes and Genetically
  Engineered Organisms
• An New Understanding of Human Origins and the
  Diversity of Human Populations (e.g., where we
  come from)
• New Understanding of the Evolutionary
  Relationships Between Organisms (e.g., sequence
  of mammalian genomes, including mouse, human,
  dog, cat,chimpanzee)
• Ability to Sequence the Genomes of Extinct
  Organisms
• New Ethical Issues in How Far We Should Go in
  Using Genetic Engineering and Mammalian
  Reprodcutive Technologies

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Genetic Engineering Technology Has Led to Many
New Legal and Ethical Issues-A Review

• Patenting Genes, Cells, Living Organisms?
• Regulating Experimentation on DNA, Cells,
  Transgenic Organisms (GMOs)?
• Regulating the Release of Genetically Modified
  Organisms into the Environment?
• Labeling of Genetically Modified Foods?
• Genetic Testing DNA Databases, Newborn Genetic
  Screening, Genetic Privacy, Involuntary or
  Voluntary Testing?
• Genetic Discrimination?
• Genetic Enhancement and Eugenics Right to
  Enhance Your Child?
• Gender Selection and Prenatal Screening of
  Genetic Diseases?
• Gene Therapy Correcting Human Genetic Diseases?
• Human Cloning and Genetic Improvement?
• Gene Testing Companies (e.g., 23andMe)
  Liability?
• Synthetic Genomes Constructing New Organisms?

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The Origins of Genetic Engineering How It All
Began 1973
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Some Applications of Genetic Engineering
Discussed To Date
Basic Sciences
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5.
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Genomics
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2.
Applied Uses
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Basic Genetic Engineering Uses..

• Isolating Genes
• Studying Basic Cellular Processes (e.g., Glow
  Protein in Jellyfish)
• Studying the Structure and Function of Genes
• What Do They Do?
• Where Are They Expressed?
• What Are There Switches?
• What is Their Structure? (e.g., intron discovery)
• Studying the Structure and Function of Proteins
  Encoded by Cloned Genes
• Sequencing Genomes
• Identifying Genes
• Understanding the Evolution of Genes,
  Chromosomes, Genomes, and Populations
• Understanding Evolutionary Processes
• Studying the Origins of Cellular Systems
• Synthetic Biology-Minimal Number of Genes to Make
  a Cell

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e.g., Cloning the Jellyfish Green Fluorescence
Protein (GFP) Gene, Dissecting GFP Structure,
Using GFP to Mark Cell Processes
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A Few Applications of Genetic Engineering
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Some Applications of Genetic Engineering
Basic Sciences
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1.
5.
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4.
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3.
2.
Applied Uses
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Some Applied Uses of Genetic Engineering

• Recombinant Nucleic Acids (DNA RNA)
• DNA Fingerprinting Probes/Templates-Forensics
• DNA Probes/Templates-Genetic Disease Diagnosis,
  Paternity, Infectious Disease Pathogens,Ancestry,
  Personal Genomics (SNPs)
• DNA Computers!
• Gene Genome Sequencing
• PCR (Need Clone Sequence First!)
• Anti-Sense/RNAi Drugs (Humans)
• Gene Therapy Vectors (Humans)
• Recombinant Viruses
• Gene Therapy Vehicles
• Vaccines
• Recombinant Microbes (Bacteria Fungi/ Molds)
• Biofactories/Metabolic Engineering- Synthesis of
  Industrial Molecules
• Drug Production- Human Proteins Antibiotics
• Enzymes Protein Engineering- Food Industrial
  Application
• Waste Remediation
• Transgenic Animals Humans
• Disease Models/Functions of Human Genes (Mouse)
• Drug Production (Whole Animals Cells)

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Genetic Engineering Applications Using Bacteria
E. Coli is the Workhorse
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Bacteria Have Many Uses and Cause Much Human
Suffering
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Expression Vectors Are Used To Make Recombinant
Proteins In Bacterial Cells
cDNA or Synthetic Coding Sequence
Foreign Protein or Drug
What Switches? Terminators? Codon Usage (For
Synthetic Genes)?
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Why E. coli ?
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Recombinant Drugs Made In Bacteria And Other Host
Systems To Treat Human Diseases
Mammalian Cells Why?
E. coli
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Regulation of Sugar Metabolism by Insulin..a
Review
One Insulin Gene-One Insulin Protein Processed
After Translation
x
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Manufacturing Recombinant Human Insulin-A Review
1. Cloning Synthetic cDNAs
3. Purification
4. FDA Approval Sale
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Must Have FDA Approval of Drug Production Process
Drug Sale and Use
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Bacterial Diseases That Are Vaccine Targets
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Viral Diseases That Are Vaccine Targets
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In 1776 George Washington Lost 1,000 Men to
Battle And 100,000 Men to Smallpox! Washington
Had His Army Innoculated With a Small Amount of
Fluid From a Smallpox Victim and the Smallpox
Rate Went Down Smallpox Was One of the First
Biological Warfare Agents-Having Been Used For
Centuries Responsible For 300-500 MILLION Deaths
in the 20th Century Eradicated From the Face of
the Earth in 1980 By Destroying All Known Viral
Stocks-Only Disease Known To Be Completely
Eradicated
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Edward Jenner Using Cowpox to Vaccinate a Child
Against Smallpox
1797
Vaccine From Vacca or Cow
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Immune System Fighting Infection at a Wound Site
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Phagocyte Destroying Bacteria in a Wound
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Immune System B-Cells Secrete Antibodies to
Specific Antigens
B-Cell Making A Specific Antibody
Variable Region Unique to Each Antibody
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Immunoglobulin Genes Are Rearranged In Genome
After Conception During B-Cell
Development-Arrangement of Genes in Sperm and Egg
Differ From Same Genes in Plasma B-Cells
144,000 Combinations of H-Chains X 144,000 Combina
tions of L-Chains 21 BILLION POSSIBLE
Antibodies Being Produced BEFORE Birth!
Specific Antibody
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Importance Of Vaccines Antibiotics in Combating
Infectious Diseases
Polio
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Genomics Can Provide Valuable Information About
Pathogen Protein Targets For Vaccine Production
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Comparison of Pathogenic and Non-Pathogenic E.
coli Genomes
E. coli O157H7 was first recognized as a
foodborne pathogen in 1982 during an
investigation into an outbreak of hemorrhagic
colitis (bloody diarrhea) associated with
consumption of contaminated hamburgers (Riley, et
al., 1983). The following year, Shiga toxin
(Stx), produced by the then little-known E. coli
O157H7, was identified as the real culprit.
75,000 Illnesses 650 Deaths Due To E.
coli H0157 72 Million Illnesses 5,000
Deaths Due to All Foodborne Diseases In US!!! A
BIG PROBLEM
http//www.cdc.gov/ncidod/eid/Vol5no5/mead.htm
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Using PCR To Detect Pathogenic and Non-Pathogenic
E. coli Strains
Need Knowledge Of DNA Sequences
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Using DNA Sequencing To Identify Bacterial
Other Pathogens (e.g., Viral)
META Genomics For the Discovery of Pathogenic
Bacteria New Antibiotic-Producing Bacteria
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Metagenomics Sequences the Genomes of MANY
ORGANISMS AT ONE TIME and Sorts Out Their
Identity Using Bioinformatics and GeneBank
Databases - Its A Powerful Organism and Gene
Discovery Tool
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Using Genetic Engineering To Make Vaccines
Clone Pathogenic Antigen Gene in E. Coli or Other
Host (e.g., Yeast, Virus) And Synthesize Large
Amounts of Antigen
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Recombinant Vaccines Are Being Developed
ToCombat Many Pathogens
HIV Destroying T-Cells
But a Vaccine To The AIDS Virus Remains Elusive!!
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Making Cancer Vaccines-Checking Abnormal Cell
Division
Normal Cell Cycle
Normal Cell Division
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Check Points Controlling Cell Division
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Mutations in Check Point Genes/Proteins Lead To
Cancer
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Recombinant Vaccines Are Being Developed To Fight
Cancer
Cancer Cell Being Destroyed By T-Cell Containing
Cancer-Cell-Specific Antibody
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Using Herceptin to Treat Breast Cancer
VaccineTarget
Trastuzumab or Herceptin
Dr. Dennis Slamon, UCLA Jonsson Cancer Treatment
Center
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Antibiotic Resistance Is Also A Major Problem in
Combating Pathogens
Methicillin Resistant Staphlococcus aureus MRSA!!
Scientific American, May, 2001
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A ReviewHow Do Antibiotics Kill Bacterial
Cells?
Penicillins
By Inhibiting Basic Microbial Cell Processes
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Genetic Engineering Can Be Used To Make
Better/More Effective Antibiotics
By Modifying Pathways Leading to Antibiotics In
Bacterial Cells..But Need To Know Genes/Proteins
in Pathway By Finding Their Targets In
Pathogens As Well
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Metagenomics Sequences the Genomes of MANY
ORGANISMS AT ONE TIME and Sorts Out Their
Identity Using Bioinformatics and GeneBank
Databases - Its a Powerful Tool For Discovering
Organisms That Make New Antibiotics
And By Finding Targets For New Drugs In
Specific Pathogens As Well
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Bacteria Other Microbes Are the Source Of Many
Different Products
Specific Proteins and/or Metabolic Pathways Can
Be Improved and/or Manipulated By Recombinant DNA!
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Recombinant Chymosin Is Used To Make Cheese
Chymosin (Rennin) Acts On Milk Proteins To
Coagulate Milk ? Cheese
Is Cheese A GMO?
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Chymosin In Cheese Making

• 80-90 of Cheeses Are Made With Recombinant
  Chymosin (a Protease)
• Approved For Use In Cheese Making By FDA - 1992
• Not Different From Non-Recombinant Chymosin-
• ?GRAS- Generally Regarded As Safe No
  Labeling Needed- Because Not An Additive Not
  Different From Non-Recombinant Chymosin!!

Is Cheese Made Using Recombinant Chymosin a GMO?
Industry Adds Claim That Recombinant Chymosin is
Kosher Vegetarian
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FDA Approval of Cheese Made With Recombinant
Chymosin
Extraordinary precautions were taken before
chymosin, made by recombinant DNA technology, was
marketed. Regulators ensured that no toxins of
any kind had been introduced and that no live
recombinant organisms were present. Indeed, the
product contained nothing but pure chymosin.
Cheese made with it is completely
indistinguishable from that produced with animal
rennet. In any case, chymosin itself is degraded
during cheese making and none is left in the
finished product. Today, in North America, over
80 percent of all cheese is made using chymosin
produced by recombinant DNA technology. Cheese
makers no longer have to worry about a shortage
of calf stomachs and turophiles can satisfy their
critical tastebuds. Thanks to biotechnology they
can "say cheese" and smile.
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Protein Engineering- Evolution In A Test Tube!!
Making Enzymes Not in Nature That Work More
Efficiently Using Recombinant DNA!
Novel Enzymes Other Proteins!
e.g., a More Effective Chymosin!
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Bacteria Produce Many Useful Metabolites, or
Small Molecules,That Can Be Engineered
Optimize Using Genetic Engineering
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Metabolites Are Produced By Cellular Pathways
That Use Specific Enzymes and Genes To
Synthesize Specific Small Molecules
Butanol or Ethanol
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Bacterial Metabolic Pathways Can Be Engineered To
Optimize Production Of Novel Industrial Products
These Pathways Can Be Optimized /Or Changed By
Adding Genes On Plasmids That Encode Novel Enzymes
Gene Shuffling Protein Evolution - Protein
Engineering
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Engineering E.coli Pathways To Make BioFuel
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Engineering E.coli To Synthesize Indigo- The
Major Blue Dye For Jeans Other Clothes Uses
E. coli Gene
Pseudomonas Gene
200M/Year Industry Indigo Previously Obtained
From Plants!
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Pollutants In Environment
Bioremediation
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Bacteria Can Be Engineered To Have Novel
Degradative Pathways For Bioremediation
Herbicide
Plasmids
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Bacteria Can Be Engineered To Degrade Several
Different Toxic Compounds
Pseudomonas
A Landmark Decision

Chakrabarty US Patent 4,259,444 1981 Genetically
Engineered Microorganisms Are Inventions
Life Can Be Patented !
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Bacteria Can Be Engineered To DegradeBiomass
Waste-Containing Cellulose (e.g., paper)
Green Waste!
Energy for Bacteria

• Energy
• For Us!!

Agriculture, Timber Processing, Human Activities
e.g., Plants Left Over From Harvests, Animal
Manure With Grasses, Municipal Water Paper,
Cotton Leftovers, Hay, Etc.
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Using Bacteria to Deliver Protein to Your Body to
Treat Disease
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Glick Molecular Biotechnology 3ed.
Using Bacteria to Deliver Protein to Your Body to
Treat Disease-Proof of Principle
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Using Engineered Bacteria to Fight Tooth Decay
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Genetic Engineering Yeasts
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Using Yeasts as Factories and Catalysts
Yeast
Eukaryote 12 Mb Genome 6,000 Genes
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Using Yeast to Make Recombinant Proteins
What Are the Advantages Over Bacteria?
Vectors? Switches?
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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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Anaerobic Fermentation By Yeasts
Anaerobic
Bread Rising
Aerobic
Champagnes
ATP ? Energy!
Vinegar (Other Microbes)
Alcoholic Beverages
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Yeasts Could Be Genetically Engineered to Enhance
Alcohol Production
Enhance Production, Alter Flavors, Remove Off
Flavors!
Havent Yet --Why?
What is a YAC?