Genetic Engineering
Gene Analysis: How?
•Humans have 6 billion DNA bases on 46 chromosomes
•Each chromosome contains between 60 million and 360 million bp’s
•Genes may only be a few thousand bases long
How do we find them and identify them?
Fundamental Tools
• Restriction Enzymes
• Ligases
• Polymerases
• The 3 above enzymes are used in Recombinant DNA Technology (1970’s)
Recurrent Themes
1. “The new tools of genetic analysis emerged from a knowledge of DNA structure and function” (Hartwell et al, 2000)
e.g. complementary base pairing is basis for hybridization techniques (labeled DNA probes)
2. “the speed, sensitivity, and accuracy of the new tools make it possible to answer questions that were impossible to resolve just a short time ago”(Hartwell et al, 2000).
e.g. Using DNA technology, it was recently discovered that HIV doesn’t become latent in all cells after infection.
Uses for Recombinant DNA Technology
• Cut GIGANTIC stands of DNA into much smaller fragments
• Amplify fragments for storage and analysis
• Identify/Isolate fragments of DNA containing a desired element: gene, control, unknown
• Characterize fragments of interest by size, location and sequence
Cutting DNA with Restriction Enzymes (endonucleases)
• Each cell contains 2 sets of 3 b. bp’s• Unwound, this would stretch 2 meters• Between 35,000 and 100,000 genes• “If you could enlarge the cell nucleus to the
size of a basketball, the unwould DNA would have the diameter of fishing line and a length of 200 km.” (Hartwell et al, 2000)
History of HGP sequencing:• 1990: HGP start—15 yrs, $3b.• 1995: H. influenzae• 1997: E. coli (5 m. bp)• 1998: C. elegans (97 m. bp)• 1999: Human chromosome 22 (36
m. bp) (HG 2.8% sequenced)• 2000: D. melanogaster (180 m. bp)• 2001: working draft of H. sapiens
(2.91 b. bp)• 2003: sequence finished early
Genetic Testing: Types
Late Onset Diseases:
•Increased risk of adult onset disease. •Positive genetic tests indicate
susceptibility or risk level. •Examples: cancer, heart disease. •Exceptions (100% chance): Huntington's
disease
Newborn Screening:
•Preventative health measure •Treatments are available. •Example: congenital hypothyroidism, PKU
Types of Genetic Tests 2
Prenatal Diagnosis:•Genetic testing of a fetus with a risk
of developmental or physical disability•Examples: Down syndrome.
Carrier Identification: •Couples considering having children •Families have a history of recessive
disorders (disorders where BOTH allelesmust be damaged for the disease to manifest)
•Example: cystic fibrosis, Tay-Sachs, sickle cell
What Does a Genetic Test “Look For”?
Chromosome karyotype (cytogenetic testing):•Look for abnormal chromosome number
or abnormal chromosome shape
Sequence of gene (DNA sequencing):•Look for misspelled DNA that indicates
mutant gene
Proteins or other metabolite (biochemical testing):•Look for tell-tale by-product of genetic
disease/disorder (usually a protein)
Genetic Tests Test Different Steps in the “Flow of Genetic Information” Pathway
CHROMOSOME
DNA
GENE
DNA
DNA
A DNA
DNA
PROTEINmRNA
Genetic tests analyze DNA, RNA, chromosomes, proteins, or metabolites (by-products) to detect mutations (or by-products of mutant genes) related to a heritable disorder:
BY-PRODUCTS
CELL
?Genetic Testing for Cystic Fibrosis?
Which of the following could be used in genetic testing to determine whether an individual has cystic fibrosis?
1. DNA sequence
2. Karyotype
3. Protein function
4. All of the above
ANSWER*
Which of the following could be used in genetic testing to determine whether an individual has cystic fibrosis?
1. DNA sequence*
2. Karyotype
3. Protein function
4. All of the above
Examples of Genetic Diseases/Disorders Revealed by Different Genetic Tests
Three Types of Genetic Tests Include Analysis of:• Chromosome karyotype• DNA sequences• Protein products
Examples of Genetic Diseases/Disorders Detected by Genetic Testing: •Chromosomes: Down Syndrome, Klinefelter•DNA sequence: Cystic Fibrosis•Protein products: alpha fetoprotein
Preimplantation Testing = Preimplantation Genetic Diagnosis
Preimplantation testing is:
•performed on early embryos resulting fromin vitro fertilization to decrease the chance of a particular genetic condition occurring in the fetus.
•used by couples with a high chance of having achild with a serious genetic disorder.
•is alternative to prenatal diagnosis and terminationof affected pregnancies.
Affected embryos are not implanted in uterus.
Raises important ethical issues: When does life begin?
http://www.genetests.org/servlet
/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=wxj4&filename=/concepts/primer/
primerusesof.html
In vitro Fertilization is…
M[23+23] [23+23] [23+23] [23+23] [XY] [XX] [XY] [XX]
Y X
MOTHER(XX)
FATHER (XY)
chromosomes
In vitro fertilization occurs in a petri dish
SpermEgg
In vitro Blastocyst can be implanted in uterus and develop.
Common Bioethical ?: Are We “Playing God”?
Ex-Utero Genetic Testing
Ex utero means "outside the uterus.“
•In vitro fertilization used to make fertilized egg.
•Cells from a developing embryo are removed andused for DNA testing to see if mutant gene is carried by that embryo.
•Embryos without the mutant genes are implantedin the mother's uterus to develop into a baby.
Could this approach be used to avoid having a child that is sick with cystic fibrosis?
STOP?
Before the Baby is Born…
Three Types of Prenatal Testing
Alpha-fetoprotein Test (AFP): AFP protein is made by the fetus and measured in Mom’s blood. Too much or too little amount of AFP indicates possible problem with fetal spine development.
Prenatal Enzyme Tests: Check fetal cells for certain enzymes (proteins). A disease will occur if that enzyme is not produced. A prenatal enzyme test is done if fetus is at risk (disease runs in the family).
Ultrasound Imaging: Sound waves create an image of the baby inside the mother. Many physical problems can be detected with ultrasound, and more tests can be performed if needed.
Points to Consider: Prenatal Testing
Prenatal Diagnostic Testing:
•Requires a clinical laboratory for disease-specificprenatal diagnostic test needed.
•All prenatal test procedures are a risk to the fetus and the pregnancy
•Risk factor assessment requires informed consent and genetic counseling.
•Usually specific gene mutation(s) and/or diseases are identified in a blood relative or in carrier parent(s).
•Prenatal testing for adult-onset conditions iscontroversial.
http://www.genetests.org/servlet
/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=wxj4&filename=/concepts/primer/
primerusesof.html
Questionnaire to Assess Risk for Prenatal Patients
What information do genetic counselors need to assess the level of risk for genetic diseases for each couple and for expected child?
Questionnaire for prenatal patients is at:http://www.genetests.org/img/assessprenat.pdf
Prenatal Testing Risk Factors Include:•maternal age •family history•ethnicity•Suggestive/positive genetic marker screen •fetal ultrasound examination
Routine prenatal diagnostic tests are:•amniocentesis (“amnio”)•chorionic villus sampling (CVS)
Amniocentesis and Chorionic Villus Sampling (CVS)
“Amnio” and CVS tests check for genetic defects:
•Doctors remove some cells surrounding the fetus•Cells are treated with a special dye and
photographed through a microscope •Chromosomes are arranged into pairs by
banding patterns•Karyotype is checked for missing, extra or broken
chromosomes
Amnio and CVS are used to obtain cells for DNA analysis. If fetus has a risk of inheriting a genetic disease, the suspect gene is tested for a mutation.
http://www.genetests.org/servlet
/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=wxj4&filename=/concepts/primer/
primerusesof.html
Once the Baby is Born…
Newborn Screening Tests are Required in U.S.
Newborn Screening is required by law in the U.S. but varies state-to state (Table):
•Massachusetts requires testing for 11/13 diseases
•States (eg. Mississippi, Missouri) require testing for only 4/13 diseases
Newborn Screening Tests:•PKU•HIV•Galactosemia•MSUD•Homocystinuria•Biotinidase Deficiency
http://www.genetests.org/servlet
/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=3kel&filename=/concepts/primer/
nbscreenmap.html#theTop
•Sickle Cell Disease•Toxoplasmosis•Cystic Fibrosis•Tyrosinemia•MCAD•Congenital Adrenal Hyperplasia•Congenital Hypothyroid
Ethics, Options and Possibly Endless Decisions
Should prenatal testing be used to select features of your baby, such as hair color, body shape or intelligence?
Some cultures value boys more than girls and parents use prenatal tests to choose the sex of their child, and abort girl fetuses.
Currently, prenatal genetic testing does not tell us much more than the sex and risk of some genetic diseases. In the future, genetic testing could make endless choices available to parents.
Now we must all decide about these choices.
Genetic testing has another important use: DNA Fingerprinting is a genetic test that can be used to identify anyone and everyone!
Understanding Stem CellsUnderstanding Stem Cells
• Stem cells have the ability to divide for indefinite periods in culture to give rise to specialized cells
PotencyPotency
• Totipotent: unlimited
• Pluripotent: little limitation
• Multipotent: highly limited
Stem Cell OriginsStem Cell Origins
• hASC’s . . . Adult Stem Cells
• hEGC’s . . . Fetal Stem Cells
• hESC’s . . . Embryonic Stem Cells
• Now iPS cells
• Where do these 3 lines come from?
Applications of Stem CellsApplications of Stem Cells
• Understanding complex developmental events
• drug development and safety
• generation of cells and tissues for therapy– heart muscle transplants
– islet cells in the pancreas
Cloning and Stem CellsCloning and Stem Cells
• Immune rejection/tissue incompatibility may be overcome by SCNT (cloning)
• Using a person’s own cells to generate stem cells could eliminate rejection
All stem cells are NOT alikeAll stem cells are NOT alike
Do ASC’s have the same potential as ESC’s?
Why not just pursue hASC’s?Why not just pursue hASC’s?
• Positive aspect of ASC’s is lack of tissue rejection and ethical issues
• Negative aspects: – apparent limitations to specialization– lack of available quantities and lines– age of cells– Can this be overcome? . . . iPSCs?
More Thoughts . . . More Thoughts . . .
• Funding for SC research: pending legislation, ethics, economy, etc.
• Funding for IVF clinics (political and ethical debates)
• The pressure is on legislatively– Americasdebate.com
What is a Blastocyst?What is a Blastocyst?
A fertilized egg divides to make 2, 4, 8, etc. cells.
Within 4-5 days there are ~100 cells in a hollow ball:the blastocyst.
Inside the blastocyst, at one end, is the inner mass of cells (embryonic stem cells.)
The blastocyst develops while the fertilized egg is traveling from the ovary to the uterus, where it will implant into the uterine wall. (“pre-implantation” embryo).
blastocyst
Therapeutic & Reproductive Cloning: Therapeutic & Reproductive Cloning: The Pathways Diverge!The Pathways Diverge!
4. Grow the egg cell (with donor nucleus inside) under suitable conditions in the lab, to the blastocyst stage:
Therapeutic
Remove stem cells from blastocyst
Implant blastocyst in surrogate mother
Reproductive
Goal is to produce a human, an exact
genetic copy
5.
Goal is to use stem cells to cure diseases and
medical conditions
Therapeutic & Reproductive Cloning DivergeTherapeutic & Reproductive Cloning Diverge
Use stem cells to do research on:
organ replacements, Alzheimer’s, diabetes,
Parkinson’s, MS, spinal cord injuries, etc.
TherapeuticCloning
ReproductiveCloning
“Replace” a lost loved one, “recreate” a hero,
duplicate yourself or friend-- but they will need time to grow up, and likely will not
be under your control!
Note: Animal reproductive cloning: save endangered species, create better food supply, “duplicate” a pet
How Do We Grow Cells in a Laboratory?How Do We Grow Cells in a Laboratory?
To study cells, they must be grown outside of an individual, in an artificial environment.
To survive outside of an organism, cells must be “fed” and cared for :
1. Nutrient soup: amino acids, carbohydrates, fats, vitamins, minerals, intact proteins
Plastic dish
Cultured cells
2. Incubator: temperature must be kept within limits characteristic of organism
What Starts the Process of Differentiation?What Starts the Process of Differentiation?
The purple cells are descended from a single cell and are genetically identical to each other. They all have exactly the same DNA sequence in their chromosomes.
How can these cells have different fates?
What Starts the Process of Differentiation?What Starts the Process of Differentiation?
At the eight-cell stage, “compaction” occurs, creating an inner and outer
layer of cells.
outer cell layer
inner cell layer
These cells still have identical genomes. But their environments have changed.
Signals Can Activate Different Parts of the GenomeSignals Can Activate Different Parts of the Genome
• Cell Division: change gene expression, make more cells-- also called “proliferation”
• Differentiate (specialize): change gene expression, become a different type of cell
Differentiation
Division
F G
A
B
C
D E
A
B
C
Cells decide what to do based on chemical & physical stimuli or “signals” from
• Inside the cell (genome, cytoplasm)• Outside the cell (environment: other cells, hormones)
How To Grow Stem CellsHow To Grow Stem Cells
1
23
4
7
6
How To Grow Stem Cells
1) In vitro fertilized egg or egg made from nucleus transfer
2) Blastocyst forms (5-7 days)3) Inner stem cell mass4) Undifferentiated stem cells
cultivated in petri dish5) Red blood cells6) Nerve cells7) Muscle cells
5
Which Number Indicates the Which Number Indicates the Undifferentiated Stem Cells? Undifferentiated Stem Cells?
2 4
6
5
1
3
Which Number Indicates the Undifferentiated Stem Cells?
Which Number Indicates the Which Number Indicates the Undifferentiated Stem Cells? (3)Undifferentiated Stem Cells? (3)
2 4
6
5
1
3
Which Number Indicates the Undifferentiated Stem Cells? (3)
Applications of Human Cloning: The Stem Cell Connection
Two Major Applications of Human Cloning:
1) Reproductive Cloning: creates a human being who is genetically identical to the person being cloned
2) Therapeutic Cloning: creates stem cells with a pre-determined genetic makeup for therapeutic use
The Major Difference: • Reproductive Cloning results in a live baby• Therapeutic Cloning creates “genetically matched”
stem cells for “personalized” medical treatment
The Promise of Stem Cell ResearchStem Cell Promise: Pie in the Sky or Real-Life Treatment?
Diabetes: • Insulin-producing pancreatic cells
Heart Disease:• Heart muscle cells to replace cardiac tissue damaged by heart attack.
RegenerateNerve Cells: • Spinal cord damage• Brain disorders• Parkinson’s• Alzheimer’s disease • Stroke and epilepsy
Bone Marrow Nerve Heart Muscle Pancreas
www.nih.gov/news/stemcell/primer.htm
Parkinson’s Disease is a Target for Stem Cell Treatment
•Parkinson’s disease: Brain neurons die. •Cells can’t make neurotransmitter, dopamine.•Dopamine is required for nerve signals and movement!
Digital Anatomist Program, University of Washington, Seattle
Symptoms start mild -- a mask-like face, an inability to initiate movement -- then general stiffness, tremors, immobility -- eventually fatal
Treat Parkinson’s by transplanting healthy nerve cells into brain. The transplanted healthy nerve cells can make dopamine? Possible problems with this approach?
Fetal Cell Transplants Temporarily “Cure” Parkinson’s: Can Stem Cells Cure Brain Disorders?
Transplant Fetal Cells into Parkinson’s Brain:•Nerve cells die in localized spot in the brain•Dead nerves replaced with undifferentiated stem cells
Implant Fetal Brain Cells Into Parkinson’s Brains:•Cells secrete dopamine!•Symptoms improve!•“Cure” is temporary.
Embryonic Stem Cells:•ESC differentiate into brain cells that secrete dopamine•Nerve cells differentiated from “cloned” ESC won’t be rejected if transplanted into donor’s brain
What Can Stem Cells Really Do?
The Very Short History of Stem Cell Research…
1998: James Thompson and group from University of Wisconsin-Madison was first in world to isolate and culture in the lab human embryonic stem cells.
Five (5) human embryonic stem cell lines were established in 1998 at Univ of Wisconsin (and still exist in 2003).
How many human stem cell lines are enough?
What source(s) of human blastocysts are acceptable?
http://www.nih.gov/news/stemcell/primer.htm Shamblott, PNAS, 95: 13726-13731 (1998), Thomson, Science, 282: 1145-1147 (1998).
Bush Limits Stem Cell Research (2001)
Stem Cell Limit: President Bush (8/9/01) announced that he would limit the federal funding of embryonic stem cell research to 60 existing cell lines. (Currently fewer than 40 established stem cell lines exist).
Why are Stable Stem Cell Lines Important?
Medical Treatment:• Renewable source of replacement cells for Alzheimer's, Parkinson’s, spinal cord injury, stroke, burns, diabetes, osteo- and rheumatoid arthritis
Research and Drug Development:• Stem cell lines increase accuracy of drug development tests, reduce cost and avoid use of animals •Stem cell research to understand human development
Paralyzed Rats “Cured” by Human Stem Cells?!
John Gearhart (Johns Hopkins Univ.) and Hans Keirstead (U California, Irvine):
•Treated mice or rats with paralyzed hind legs •Injected or infused stem cells into the rat’s spinal cords•Treatment restored some movement in rat’s hind legs!!
The Two Groups Used Different Sources of Stem Cells:
•Pre-germ cells from 5- to 7-week-old aborted human embryos (Gearhart). •Human embryonic stem cells from embryos donated by IVF couples “informed consent” (Keirstead)
http://www.wired.com/news/medtech/0,1286,50951,00.html
Therapeutic & Reproductive Cloning Follow the Same Pathway…
4. Grow the egg cell (with donor nucleus inside) under suitable conditions in the lab, to the blastocyst stage.
Egg cell (contains donor nucleus after nuclear transfer)
blastocyst
Only recently achieved for humans (1998)Not fully understood, difficult to control
Therapeutic & Reproductive Cloning: Both Start the Same Way
whyfiles.org/148clone_clash/2.html
1) Nucleus removed from egg cell.
2) Pipette picks up one nucleus
3) Nucleus to be cloned is transferred into empty egg
Your Genome Master Plan
M[23+23] [23+23] [23+23] [23+23] [XY] [XX] [XY] [XX]
Y X
SPERM ON EGG
SPERM
EGG
MOTHER(XX)
FATHER (XY)
chromosomes
OK, So I Could Clone YOU Using All the DNA In a Nucleus from One of Your Cells…Right?
Human Cell
DNA
RNA
CELL MEMBRANE - need to rupture cell membrane to get the nucleus out of cell.
NUCLEUS - stores DNA in chromosomes, and is strong enough to be physically transferred from cell to cell (without dropping chromosomes)!!
Once I get the nucleus (with its Master Plan) out of the “old” cell, what next? The nucleus needs some cytoplasm!!
CYTOPLASM
OUT With the “old” DNA…
“old” cell
DNA
NUCLEAR TRANSFER
Once I get the nucleus (with its Master Plan) out of the “old” cell, what next? The nucleus needs some cytoplasm!!
CYTOPLASM
DNA ?
IN With the “new” DNA…
“old” cell
DNA
NUCLEAR TRANSFER
The nucleus to be cloned is injected into an egg without a nucleus!
CYTOPLASM
DNA DNA
“new” cell
OLD NUCLEUSXRemove nucleus!
In With the “new” DNA…
“old” cellNUCLEAR TRANSFER
The nucleus to be cloned is injected into an empty egg without a nucleus.
DNA
“cloned” cell starts to undergo cell division
DNADNA
X
DNA
DNADNA
DNA
In With the “new” DNA…
NUCLEAR TRANSFERThe nucleus to be cloned is injected into an empty
egg without a nucleus. DNADNA
DNA
DNADNA
DNADNA
DNA
DNA
The cells continue to divide and make more genetically identical cells! These cells are not specialized!
DNADNADNA
DNADNA
DNA
The Donor Nucleus is Transferred in Human Cloning!
Donor Nucleus (from the person to be cloned)
Egg Cell is Prepared by Discarding its Nucleus
Donor Nucleus is Transferred into Egg
1)
3)
2)
[Egg Nucleus (discard egg nucleus and egg genome)]
Zygote begins cell divisions
4)
Basic Steps in Animal Cloning:First We Need an Egg Cell and a Cell from the Adult to Be Cloned
•A woman contributes an egg
•An individual contributes an adult cell
•The nucleus is removed from the egg
Basic Steps in Animal Cloning:Next We Remove the Nucleus from the Egg Cell
•A woman contributes an egg
•An individual contributes an adult cell
•The nucleus is removed from the egg
Basic Steps in Animal Cloning:Then the Nucleus from the Adult Cell is Moved into the Egg Cell
•A woman contributes an egg
•An individual contributes an adult cell
•The nucleus is removed from the egg
•The nucleus from the adult cell is transferred to the egg
Basic Steps in Animal Cloning:Then the Nucleus from the Adult Cell is Moved into the Egg Cell
•A woman contributes an egg
•An individual contributes an adult cell
•The nucleus is removed from the egg
•The nucleus from the adult cell is transferred to the egg
Animials that have been clonedAnimials that have been cloned
• Tadpole (1952) from embryonic cells (?)
• Fish (1963) China (? No English)
• Sheep (1996, 1997) *first mammal, first from SCNT
• Many more since 2000 including “Little Nicky” cat $50,000 Complete list
Hello Dolly! The First Cloned Animal: 1997!
Dolly and Dolly’s Surrogate Mother
Dolly the cloned sheep:
•Nuclei from adult sheep cells were transferred into 277 sheep eggs (without nuclei)
•“Cloned” eggs were implanted into female sheep for gestation.
•One cloned lamb born: Dolly
•Dolly is an exact genetic copy of the adult sheep that donated the nucleus!
PPL Therapeutics, Roslin Institute, Edinburgh, Scotland:www.sciam.com/explorations/030397clone/030397beards.html
Hello Dolly’s Lamb!
Dolly and her
lamb!
Does the “health” of the cloned animal depend on the “age” of the donor nucleus?
How “old” is Dolly?
Dolly was cloned in 1997 using nuclei from a 6-year old ewe. This makes Dolly either 6 or 12 years old in 2003.
PPL Therapeutics, Scotlandwww.wired.com/news/medtech/0,1286,49450,00.html
Dolly has developed arthritis in her leg joints in 2001. Why? Is Dolly “old before her time” because she was cloned from a 6-year-old ewe? (Ans: she died from lung cancer common to sheep actually)
Did Dolly get “old” DNA?
Speculated Problem Cloning with “Old” DNA?
In human cloning, an adult nucleus with 46 “adult” chromosomes is transferred into an egg cell (without a nucleus). “Adult” chromosomes are pre-programmed to express “adult” genes. The “adult” DNA must be re-programmed to start development at the beginning.
Human development: 23 chromosomes from Mom and 23 chromosomes from Dad, come together to make a complete genome of 46 chromosomes in a totipotent nucleus, in an egg, ready to develop.
Adult cell
Egg cell
Clonedcell
Adult nucleus “old” DNA
Egg nucleus “new” DNA
Cloning Animals: Can Pigs Grow People Parts? Problem of Tissue Rejection: When a human organ is transplanted into another person, it is rejected as “foreign” by recipient’s immune system.
Pigs are a possible source of human transplant organs but the pig organs are rejected as “foreign” by humans.
Four piglets born Christmas 2001 are “genetically engineered” to TURN OFF a pig gene that causes tissue rejection in humans.
Tissues grown in the Christmas piglets might be transplanted into humans without rejection because they have a rejection gene removed from the pig genome.
Christmas piglets are a first step toward engineering animals that grow organs for transplant into humans.
http://www.wired.com/news/medtech/0,1286,49450,00.html
Cloning Noah’s Ark: Saving Endangered Species
ACT http://www.advancedcell.com/
Advanced Cell Technologies
Saving Endangered Animals: "Inter-species Nuclear Transfer"
Cloning can potentially be used to preserve endangered species without the problems of breeding endangered animals in captivity.
Cloning, 2(2):79-90, 2000. http://www.advancedcell.com/animal-program.html
Noah’s “Dad”: an endangered ox
(Bos gaurus)
Noah is born!
Noah’s “Mom”:a domestic cow!
The first endangered animal, a gaur ox (Bos gaurus), was produced by inter-species nuclear transfer in 2001.
Gaur is an ox native to India, Indonesia and southeast Asia. Only ~ 36,000 gaur remain!
Cloning Noah:• Male gaur cell nuclei injected into 692 eggs from domestic cows• Cell division induced and 81 blastocysts were formed• 42 blastocysts implanted in 32 surrogate mother cows• 8 pregnancies, and 1 live birth, Noah.
Cloning Noah: Count The Numbers!
Is Noah male or female?
How was Noah’s gender determined?
Cloned kitten and surrogate Mom
Questions Raised About Animal Cloning
Major Concerns About Animal Cloning:
• Cloning process is not efficient• Is it ethical to clone animals for human use?• Are there problems when the old cytoplasm re-programs the young nucleus?
Copy Cat
HUMAN CLONING: NO LONGER FICTION,
WHAT ARE THE FACTS?
Taken from ACT website and Scientific American
Behind the Headlines: Read More About the Controversy!
Read More About the Controversy:Wired News: ACT Cloning All Hype?www.advancedcell.com/pr_11-25-2001.html
The First Human Cloned Embryo! by Advanced Cell Technology, Worcester, MA
Scientific American, U.S. News and World Report,
e-biomed: The Journal of Regenerative MedicineSunday, Nov. 25, 2001
Meet the First Cloned Human: Dr. Judson Somerville
The First Human Cloned Embryo! ?by Advanced Cell Technology, Worcester, MA, 2001
Who is Dr. Judson Somerville?
•a medical doctor, an accident left him paralyzed•Somerville met Dr. Michael D. West, (CEO of ACT), at Baylor Medical School•Somerville wanted to donate his own skin cells to a human cloning therapy that might someday allow him, and many others, to walk again.
Scientific American, 2001
Somerville’s cloned cells grew to 6-8 cell stage.
What about the 98% of the genome that is not made up of GENES? This
is today’s genetic HOT topic.
• Could it play a complex role in gene expression?
• Humans only have 23,000 genes (less than an ear of corn!)
• Complexity comes from somewhere else
• TF (switches), HOX genes, RNAi• Big areas of study today
The Role of Transcription Factors in Controlling“Tissue Specific” Gene Expression
(a) Transcription factors are proteins that control gene expression
(b) Transcription factors are specific to certain tissues or cell types (“nerve cell-specific” TFs are made ONLY in nerve cells, etc)
(c) Transcription factors can turn a gene ON in one cell type but may not in another kind of cell
(d) Transcription factors bind directly to DNA
DNA
PROTEIN
PROTEIN
DNA
Transcription Factors Required for Stem Cells to Stay Pluripotent!
Oct4 and Sox2 are TF proteins in mammalian stem cells
Oct4 and Sox2 TF are required to maintain pluripotency in developing mammalian embryonic stem cells (ESCs)
Sox2 knockout embryos die because they couldn’t differentiate. Sox2 knockout mice have both copies of the Sox2 gene deleted (knocked out).
Sox2 discovered by Robin Lovell-Badge MRC, London, UK (Knocking the Sox Off Early Mammalian Developmenthttp://www.sciencedaily.com/releases/2003/01/030101222928.htm)
Sox2 gene
Sox2 gene
deleted Sox2
deleted Sox2
DN
A
Differentiation: Know the Right Signal?!
http://gened.emc.maricopa.edu/bio/bio181/BIOBK/BioBookAnimalTS.html
Cells lining a tubuleFat cells
Muscle cells
Red blood cells
Cells lining the
small intestine
Cell types in the stomach
Nerve cells
Human body has 220 different cell types in tissues and organs!
Synthetic Chromosome = Artificial Life?
The work by Dr. Craig Venter
If you could design a new bacteria that would be helpful,
what would your bacteria
be able to do?
Dr. J. Craig Venter• Ph.D. in physiology and pharmacology• Founded The Institute for Genomic Research
at NIH - 1992• Founder and former president of Celera
Genomics - 1998– Fired in 2002
• Current president of the J. Craig Venter Institute
Cracking The Code
• The human genome sequencing race.
• Venter announced Celera would accomplish the sequencing by a target date.
• Government scientists stimulated to collaborate, and then finish the genome.
A Man of Controversy
• Tried and failed to patent Essential Sequence tags (ESTs).
• Goal of Celera: sequence the human genome and sell the information.
• Sequenced his own DNA, included in the Celera genome project.
Ownership of DNA
What would be the impact to science if Donald Trump owned the right to
the genome information?
Synthetic Genomics
• Co-founded by Venter in June of 2005 to develop designed organisms.
• “At Synthetic Genomics Inc., we are developing novel genomic-driven strategies to address global energy and environmental challenges.”
The Synthetic Chromosome
• Synthesis completed in January 2008
• Constructed through homologous recombination in yeast.
• 580,000 base pairs of code based upon M. genitalium
• 381 genes
• Watermarks for identification
Mycoplasma laboratorium
• Experimental results have not been officially released.
• Patent has been applied for to own the right to use and distribute these new organisms.
The potential scoop from The Guardian
• The original bacterium had a fifth of its DNA removed
• It was then able to live successfully with the synthetic chromosome in place.
• Organism still relied upon the intracellular machinery already present to carry out tasks such as replication and metabolism
Ethical Questions
• Is it still artificial life if the new organism relies on the replication machinery of the original cell?
• Is it ethical to do this work in a parasitic organism?
Patent - Should this be allowed?
• Is it right to patent a species of bacteria, synthetic or not?
• Is it right to patent DNA sequences?
• What if the bacteria can produce an alternative biofuel - Venter’s ultimate goal?