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?