Genetic Engineering

1. Genetic Engineering

2. 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?

3. Fundamental Tools • Restriction Enzymes • Ligases • Polymerases • The 3 above enzymes are used in Recombinant DNA Technology (1970’s)

4. 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)

5. 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.

6. 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

7. 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)

8. 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

9. 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

10. 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 alleles • must be damaged for the disease to manifest) • Example: cystic fibrosis, Tay-Sachs, sickle cell

11. 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)

12. DNA DNA CHROMOSOME G E N E A DNA DNA Genetic Tests Test Different Steps in the “Flow of Genetic Information” Pathway 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 DNA CELL mRNA PROTEIN

13. ?Genetic Testing for Cystic Fibrosis? Which of the following could be used in genetic testing to determine whether an individual has cystic fibrosis? DNA sequence Karyotype Protein function All of the above

14. ANSWER* Which of the following could be used in genetic testing to determine whether an individual has cystic fibrosis? DNA sequence* Karyotype Protein function All of the above

15. 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

16. Preimplantation Testing = Preimplantation Genetic Diagnosis • Preimplantation testing is: • performed on early embryos resulting from • in vitro fertilization to decrease the chance of a particular genetic condition occurring in the fetus. • used by couples with a high chance of having a • child with a serious genetic disorder. • is alternative to prenatal diagnosis and termination • of 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

17. In vitro Fertilization is… MOTHER (XX) FATHER (XY) In vitro fertilization occurs in a petri dish Y X chromosomes Egg Sperm In vitro Blastocyst can be implanted in uterus and develop. [23+23][23+23][23+23][23+23] [XY][XX][XY] [XX] M

18. Common Bioethical ?: Are We “Playing God”?

19. 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 and • used for DNA testing to see if mutant gene is carried by that embryo. • Embryos without the mutant genes are implanted • in 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

20. Before the Baby is Born…

21. 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.

22. Points to Consider: Prenatal Testing • Prenatal Diagnostic Testing: • Requires a clinical laboratory for disease-specific • prenatal 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 is • controversial. http://www.genetests.org/servlet/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=wxj4&filename=/concepts/primer/primerusesof.html

23. 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? • 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) Questionnaire for prenatal patients is at: http://www.genetests.org/img/assessprenat.pdf

24. 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

25. Once the Baby is Born…

26. 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 • Sickle Cell Disease • Toxoplasmosis • Cystic Fibrosis • Tyrosinemia • MCAD • Congenital Adrenal Hyperplasia • Congenital Hypothyroid http://www.genetests.org/servlet/access?id=8888892&key=J7yF5J0cK5SoH&fcn=y&fw=3kel&filename=/concepts/primer/nbscreenmap.html#theTop

27. 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!

28. Understanding Stem Cells • Stem cells have the ability to divide for indefinite periods in culture to give rise to specialized cells

29. Potency • Totipotent: unlimited • Pluripotent: little limitation • Multipotent: highly limited

30. Stem 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?

31. Applications 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

32. Cloning 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

33. All stem cells are NOT alike Do ASC’s have the same potential as ESC’s? 2006 answer: NO . . . 2009 Maybe?

34. 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?

35. 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

36. 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

37. 5. Therapeutic Reproductive Remove stem cells from blastocyst Implant blastocyst in surrogate mother Goal is to produce a human, an exact genetic copy Goal is to use stem cells to cure diseases and medical conditions Therapeutic & Reproductive Cloning: The Pathways Diverge! 4. Grow the egg cell (with donor nucleus inside) under suitable conditions in the lab, to the blastocyst stage:

38. Therapeutic & Reproductive Cloning Diverge TherapeuticCloning ReproductiveCloning Use stem cells to do research on: organ replacements, Alzheimer’s, diabetes, Parkinson’s, MS, spinal cord injuries, etc. “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

39. 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 2. Incubator: temperature must be kept within limits characteristic of organism Cultured cells Plastic dish

40. 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?

41. 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.

42. A Division B • Cell Division:change gene expression, make more cells-- also called “proliferation” • Differentiate (specialize): change gene expression, become a different type of cell C G F A Differentiation B C E D Signals Can Activate Different Parts of the Genome • 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)

43. How To Grow Stem Cells How To Grow Stem Cells 1 5 3 4 2 • In vitro fertilized egg or egg made from nucleus transfer • Blastocyst forms (5-7 days) • Inner stem cell mass • Undifferentiated stem cells cultivated in petri dish • Red blood cells • Nerve cells • Muscle cells 6 7

44. Which Number Indicates the Undifferentiated Stem Cells? Which Number Indicates the Undifferentiated Stem Cells? 1 4 2 3 5 6

45. Which Number Indicates the Undifferentiated Stem Cells? (3) Which Number Indicates the Undifferentiated Stem Cells? (3) 1 4 2 3 5 6

46. Applications of Human Cloning: The Stem Cell Connection • Two Major Applications of Human Cloning: • 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

47. The Promise of Stem Cell Research Stem 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. • Regenerate • Nerve 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

48. 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! 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? Digital Anatomist Program, University of Washington, Seattle

49. 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

50. 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).