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  • Bubble boy (SCID) popularized in the 1970s of a young boy in Texas who survived to the age of 12 in a sealed environment. ADA defect: deoxyadenosine produced in response to DNA degradation. Is converted to deoxynucleotides, which inhibit white blood cell proliferation. ADA converts deoxyadenosine to deoxyinosine. Gamma chain is linked to IL-2 receptor, required for T-cell maturation from bone stem cells.
  • Humans have improved on nature’s support of plant and animal growth since they discovered that the loosening of soil and planting of seeds could result in new plants. Even prior to that discovery, they probably aided plant growth by keeping animals away from plans until they yielded fruit or other plant parts which were edible by humans.
  • History documents the domestication of the dog, horse, sheep, goats, ox, and other animals thousands of years ago. Improvement by selection soon followed. Improvement by selection means picking the best plants and animals for producing the next generation. As people bought, sold, bartered, and traded, they were able to get animals which had desirable characteristics, such as speed, gentleness, strength, color, size, milk production, and the like. By mating animals with characteristics that humans preferred, the offspring of those animals would tend to imitate the characteristics of the parents and further intensify the desired characteristics. By accident, the owner was practicing selective breeding or the selection of parents to get desirable characteristics in the offspring. The chariot armies of the Egyptians and Romans; the might of the Chinese emperors; the speed of the invading barbarians into northern Europe; the strength of mounts carrying armored knights into battle; and the evasive Arabians of the desert’ all provide convincing testimony to early successes at breeding horses for specific purposes.
  • This slide shows the basic steps of plant tissue culture. Some plant part is placed is on a defined culture media. That media induces the the tissue to develop callus. Callus is an undifferentiated mass of cells. These cells then grow into plant shoots, which are later rooted. The small seedling will then grown into a mature, seed-producing plant. When developing transgenic plants, the transformation cassette is introduced into that plant part that can be induced to grow new plants.
  • In 1988, California scientists made the first outdoor tests of ice-minus. Ice-minus is bacteria that was genetically altered to retard frost formation on plant leaves. Synthetic chemicals are now available to protect fruit crops when temperatures fall 4 to 6 degrees below what would normally damage the fruiting process. Similarly in 1988, genetically altered bacteria was injected into elm tress in an effort to control the deadly Dutch Elm disease. Further, bacteria was genetically engineered so they turn a brilliant shade of blue in the presence of a compound called X-Gal. Such bacteria can be easily detected and traced in experimental or real-life situations. The ability to so mark organisms has made biotechnology a safer and more manageable enterprise. In animal science, the hormone bovine somatotropin has long been know for its stimulation of increased milk production in cows. However, it was not available for commercial use until bacteria was altered to produce the hormone. Another example of hormone production by genetically altered bacteria is an animal hormone called porcine somatotropin, which increases meat production in swine. In plant science, Roundup and Liberty Ready Corn and Soybeans and BT Corn are examples of where genes have been altered enabling herbicide resistance and insect control through biotechnology.
  • Transcript

    • 1. Genetics III : Genetics and Biotechnology
    • 2. What is biotechnology ? Teddy
    • 3. What is biotechnology ? United Nations Convention on Biological Diversity : "Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."
    • 4. Biotechnology : past and present
    • 5.
    • 6. Biotechnology
      • Genetics counseling and diagnosis
      • Gene therapy
      • Human genome project
      • Plant and animal cloning
      • Genetic engineering and GMO
      • DNA fingerprinting
    • 7. Who ? Prof. Tsui Lap Chee ? Profession ? Vice-Chancellor of HKU Academic contributions? identified the defective gene, namely Cystic fibrosis transmembrane conductance regulator (CFTR), that causes cystic fibrosis
    • 8.
      • Cystic fibrosis
      • Cystic fibrosis affects 1 in every 3,000 births
      • Cause: an inherited autosomal recessive gene
      • Symptoms and effects:
      • Very sticky mucus produced in lungs and pancreas causing lung infections and inability to absorb food from the gut properly.
    • 9. Types of genetic testing
      • Prenatal or post natal counselling
    • 10.
      • Case study: genetics counselor
    • 11. What is his chance of having offspring with CF diseases? (CF gene is a recessive allele.)
      • The man need to determine if he is a carrier.
        • If he is a carrier
          • If her wife doesn't have family history of CF disease, the chance of their offspring having the disease is rather low.
          • If her wife has family history of CF disease, she need to know whether she is a carrier.
            • If she is a carrier, the chance of having a CF offspring is 25%. (Aa x Aa)
    • 12. What test would you advice him to do in order to determine whether he is a carrier of the CF gene?
      • He should take a CF carrier test (a blood test) to determine if he gets the abnormal gene that causes CF.
    • 13. How can genetic counseling or genetic diagnosing help them to get better prepared for the birth of the child?
      • They can make an informed decision about pregnancy.
      • undergo an invasive diagnostic procedure during pregnancy to determine whether their fetus is affected
      • choose pregnancy termination to prevent birth of an affected infant.
      • opt for preimplantation genetic diagnosis and the selective transfer of embryos without CF; but this requires in vitro fertilization
    • 14.
      • 2 . Diagnostic – to make or confirm diagnosis, generally where person already exhibits signs or symptoms of a disorder
      • Genetic disorders that can be diagnosed: e.g. Thalassemia, diabetes, leukemia, breast cancer.....
    • 15.
    • 16. Gene therapy - definition
      • The specific treatment of genetic disease
      • Modifying the expression of a person’s genes towards a therapeutic goal
    • 17.
    • 18. The methodology of gene therapy
      • Identification of defective gene
      • Genetic engineering using normal/functional gene
      • Transfer of this material into target cells (gene delivery)
      • Integration of DNA into target cell genome
      • Correction of defect
    • 19. Contributions of gene therapy: Good news : Promising advances during the last two decades in recombinant DNA technology . Bad news : Efficacy in any gene therapy protocol not definitive. 1. Shortcomings in all current gene transfer vectors. 2. Inadequate understanding of biological interactions of vector and host.
    • 20. Contributions of gene therapy 1. Cancer
      • Enhancement of immunological responses to the tumour
      • Modification of anti-oncogenes
      • Manufacture of anti-cancer factors
    • 21. Contributions of gene therapy 2. Infectious Diseases: HIV
      • Direct interference with viral processes
      • Genetic vaccination with HIV proteins
    • 22. 3. Inherited/monogenic disorders: Parkinson’s disease
    • 23. Case study
      • Treatment of S evere C ombined I mmuno D eficiency (SCID)
      • Genetic defects cause decreased T and B cells.
      • Affects 1-75,000 births.
      • Mostly males (most common form is X-linked)
      • Cause: ADA (adenine deaminase) deficiency
      • Treatment options
        • Germfree environment
        • Bone marrow transplant
        • Gene therapy
    • 24. 10-3-02 : France and US (FDA) halted SCID gene therapy due to leukemia-like side effects in one child. Not clear whether this is related to the gene therapy itself. 1/14/03: FDA suspended 30 gene therapy trials using retrovirus vectors due to another case of leukemia. clinical trials results : Detectable levels of T cells containing the introduced gene were found in the blood within 30 and 60 days, respectively, and their numbers increased progressively until normal levels were reached. After 3 months, the patients were also able to make antibodies in response to vaccination against diphtheria, tetanus, and pertussis.
    • 25.
    • 26. Which are the 3 biggest science projects in the history of mankind?
    • 27. 1942-45 Manhattan project
    • 28. 1967-72 Apollo project
    • 29. 1990-2003 Human Genome Project
    • 30. Human Genome Project
      • Project goals :
      • identify all the approximately 30,000-35,000 genes in human DNA,
      • determine the sequences of the 3 billion chemical base pairs that make up human DNA,  
    • 31. Human Genome Project
      • Milestones :
      • June 2000 completion of a working draft of the entire human genome
      • February 2001 analyses of the working draft are published in Nature
      • 2003 completed
    • 32. Some results Take a look at human chromosome: Home page of the human genome project
    • 33. Characteristics / number of genes • The total number of genes is estimated at 30,000 to 35,000 much lower than previous estimates.  • Almost all (99.9%) nucleotide bases are exactly the same in all people. (Side track questions: any 2 chimpanzees are more dislike than any 2 human, why?)  • The functions are unknown for over 50% of discovered genes.
    • 34. How It's Arranged?
      • • Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231).
      • Genes appear to be concentrated in random areas along the genome, with vast expanses of noncoding DNA between.
      • Less than 2% of the genome codes for proteins.
      • Repeated sequences that do not code for proteins ("junk DNA") make up at least 50% of the human genome.
    • 35. Applications
      • • Over 30 genes have been pinpointed and associated with some diseases (e.g. breast cancer, muscle disease, deafness, and blindness).
      • Additionally, finding the DNA sequences underlying such common diseases provide focused targets for the development of effective new therapies.
      • In the past, researchers studied one or a few genes at a time. With whole-genome sequences known, they can approach questions systematically and on a grand scale.
    • 36. Discussion Questions 1. If the genome study obtain a franchise,how can we protect the right of the poor?   2. Now, the main area of application of the genome study is for medical purpose. However, with the same technique, it is also possible to manipulate genes for other purposes, such as increasing the longevity of life and I.Q. If then, is it ethical to create such "perfect" human beings?   3. Is it ethical to change the genes of other plants and animals? What are the risks behind?   4. It may be possible to prepare a tailor-made health-care program for each individual according his or her genes. Will the privacy of individuals be respected?
    • 37. Improving Plant and Animal Performance
    • 38. Improvement by Selection
      • Soon followed domestication of the dog, horse, sheep, goat, ox and other animals thousands of years ago
      • Mating plants and animals with desirable traits resulted in selective breeding
    • 39.
        • Selective Breeding - selecting animals with desired characteristics to produce next generation (domestic animals: horses, cats, farm animals, crop plants)
    • 40.
          • 1. Inbreeding - continued breeding of individuals with similar characteristics. Can create serious problems (bringing together 2 recessive alleles)
      Albinism present in Hopi Indians. Only albino children are produced by albino parents. Two phenotypically normal individuals produce albino and non-albino children.
    • 41.
          • 2. Hybridization - crossing dissimilar individuals to bring together the best of both organisms. (e.g. disease resistance and food-producing capacity)
      Hereford, bred for meat production
    • 42. Changes in whole sets of chromosomes
      • Polyploidy is the possession of more than 2 complete sets of chromosomes.
      • e.g. triploid means 3 sets; tetraploid means 4 sets.
      • Formation of tetraploid offspring :
      • fertilization of diploid gametes or
      • whole set of chromosomes doubles after fertilization
    • 43.
    • 44.
    • 45. Plant cloning: plant Tissue Culture A Requirement for Transgenic Development A plant part Is cultured Callus grows Shoots develop Shoots are rooted; plant grows to maturity
    • 46.
        • Cloning - a member of a population of genetically identical cells produced from a single cell
      The adult sheep is Dolly, the first mammal cloned from an adult cell. The lamb is Dolly’s offspring, called Bonnie.
    • 47. How Dolly was created
    • 48. PROs
      • 1 . Producing a greater understanding of the cause of miscarriages
      • 2 . Generation of genetically modified animal organs
      • 3. Preventing child suffering heredity
      • 4. Cure damaged nervous system
    • 49. CONs
      • 1. Reducing genetic variability
      • 2. Interfering with natural evolution
      • 3.Risk of disease transfer
      • 4 . Genetic tailoring of offspring
      • 5. Detrimental effect on familial relationships
    • 50. Genetics and Genetic Engineering
    • 51. Genetic Engineering
      • technology involved in removing, modifying, or adding genes to a DNA molecule
    • 52. Cloning DNA into a Plasmid Both plasmid and foreign DNA have sticky Eco R I ends Insertion into E. coli (transformation) Agar plates contain antibiotic. Grow at 37 °C Place 1 colony in liquid media + antibiotic. Grow at 37 °C Purify Plasmid DNA (Billions of copies)
    • 53. Restriction Endonucleases
      • Restriction endonucleases cut DNA
      • Present in bacteria
      • Cut at sequence specific sites
        • Usually 4 or 6 base pairs long
      • Bacteria use them to destroy ‘foreign’ DNA
      • Restriction enzymes can be purified and are used in genetic engineering studies
    • 54. Restriction Endonucleases
      • Example Restriction enzymes
        • Eco R I ( E . co li R estriction Endonuclease I )
        • Stu I ( S treptomyces tu bercidicus I)
      GAATTC CTTAAG 5’ 5’ 3’ 3’ Sticky Ended Blunt Ended AGGCCT TCCGGA 5’ 5’ 3’ 3’ Eco R I Stu I
    • 55. Ligation of DNA Eco R I OH 3’ 5’ PO 4 PO 4 5’ 3’ OH T4 DNA Ligase Stu I Circular DNA Eco R I G CTTAA AATTC G
    • 56. Cloning DNA into Plasmids
      • Bacteria have a circular DNA genome
        • 5 to 10 million base pairs (bp) in size
      • Many bacteria also contain plasmids
        • Small circular DNA molecules, ~3,000 to 50,000 bp
        • Note : The bacterial genome is not a plasmid
      • Plasmids contain ‘extra’ genes which are often vital for the survival of the bacterium
        • Nutrient metabolism, antibiotic resistance
      • Plasmids can be used as vectors in which foreign DNA can be ligated (cloned)
    • 57. Transfer the vector to the host
        • bacteria: electroporation
        • Animal cells or plant cells: by infecting with a virus
        • - bacteriophage
        • - adenovirus
        • - retrovirus
        • (c) Gene therapy: using liposomes
    • 58. Screening
      • Identification of successful transformed cells
      • E.g. using antibiotic resistant gene as the marker
    • 59. Genetically modified (GM) food
    • 60. Improving Plants and Animals
      • 1988- first use of ice-minusfor Dutch Elm Disease
      • Long life tomatoes
      • BT Corn
      • Nitrogen fixing crops
    • 61. Polymerase Chain Reaction (PCR)
      • Technique devised in 1983 to amplify small amounts of DNA
      • Can be performed on DNA from a single cell
      • - cigarette butt, a licked stamp, root of a single hair, 1/50,000 a drop of blood (0.1 microliters)
      • The amplified DNA can then be used to:
      • - identify a suspect or victim
      • -determine an individual’s sex
      • -determine species (if not human)
      • -parentage test
    • 62. PCR to Amplify a Person’s DNA
      • Steps Involved:
      • Isolate VNTRs from a person’s DNA using restriction enzymes
      • Design primers – short segments of synthetic DNA that are complementary to DNA on either side of the VNTR regions
    • 63.
      • Add vast excess of the primers and heat mixture to 75 o C
      • This causes DNA strands to separate by breaking hydrogen bonds between bases
    • 64. 4. Cool to 15 o C. Primers hydrogen bond ( anneal ) to complementary strands 5. Add DNA polymerase and all four types of nucleotides. The polymerase (enzyme used in DNA replication) will fill in the rest of the two strands.
    • 65. You now have two identical copies of the DNA you started with. 6. Repeat steps. Heat to break hydrogen bonds. Cool to anneal more primers (still there in vast excess). Allow DNA polymerase to fill in the remaining strands. Two strands of DNA become four. Etc…Etc…Etc…..
    • 66.
      • Types of DNA probes:
      • RFLPs (restriction sites)
      • Repeated DNA
        • Minisatellites (VNTRs = variable number tandem repeats)
          • Repeated units of 5 to several 10 bp
        • Microsatellites (STRs = short tandem repeats)
          • Repeated units of 2-6 bp
          • 5’-TAATAATAATAATAATAA-3’
          • 3’-ATTATTATTATTATTATT-5’
    • 67. How to make Antisense RNA mRNA and asRNA can form RNA/RNA duplex, which actually results in formation of 20-22 nts small RNA that trigger the degradation of mRNA transcribed region 5’ control region 3’ control region mRNA transcribed region 5’ control region 3’ control region Antisense mRNA (asRNA)
    • 68.
      • DNA Sequencing
      • DNA sequencing = determining the nucleotide sequence of DNA.
      • Developed by Frederick Sanger in the 1970s.
    • 69. DNA Fingerprinting
      • Used to identify individuals by their VNTR, RFLP, or STR regions
      • Steps involved:
      • Isolate and amplify DNA if needed
      • DNA is cleaved into smaller pieces with restriction enzymes
      • DNA is separated with gel electrophoresis
    • 70.
      • DNA is transferred to a nylon membrane (blotting)
      • A radioactive primer is designed that will be complementary to unique regions (VNTR etc). Add this to nylon membrane containing DNA.
      • Wash off excess primer and hold nylon up to a photographic plate to expose. The pattern will be unique to the individual.
    • 71.
      • How to fingerprint alleged paternity using microsatellites:
      • Extract DNA from mother, baby, and alleged father.
      • Synthesize oligonucleotide microsatellite primers and label one primer with fluorescent dye (2 primers per microsatellite).
      • Amplify microsatellites using PCR from mother, baby, father.
      • Electrophores microsatellite PCR products on a DNA sequencer (w/polyacrylamide) with a flourescent size standard loaded in the same lane or capillary.
      • 3-4 different microsatellites can be multiplexed in each lane or capillary by using 3-4 different fluorescent dyes.
      • Calculate size of each microsatellite relative to size standard (this size standard also can be run in the same gel lane or capillary using a 4th or 5th colored dye).
      • Sequence at least one copy of each allele to verify allele sizes.
    • 72. Size Mother Baby “Father”
      • Paternity Analyses & Conclusions :
      • Baby and mother are expected to share on allele, and the baby and father the other allele.
      • If baby and father do not share a common allele, the “father” is not the father.
      • If the baby and father do share a common allele, paternity is possible, but not proven, because other men in the population also carry the allele at some frequency.
    • 73. Forensics • identify potential suspects whose DNA may match evidence left at crime scenes • exonerate persons wrongly accused of crimes • identify crime and catastrophe victims
    • 74. Clearly, suspect one is the match….. If all RFLP and STR regions are considered, there is a one in 3.4 billion chance of error. This means there may be one other person on the planet that would be too similar to tell the difference. If other VNTR regions are also considered, the chances of error go way, way down