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2011-07-16 13:59:46

Applications of Molecular and Cell Biology
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  1. Totipotency
    Zygotic stem cells which have ability to differentiate into any cell type to form whole organisms including extra-embryonic membranes and so are also pluripotent and multipotent
  2. Pluripotency
    Embryonic stem cells which have ability to differentiate into almost any cell type to form any organ BUT not those of extra embryonic membranes or type of cell and so are not totipotent but multipotent
  3. Multipotency
    • Blood stem cells which have ability to differentiate into a limited range of cell type and so are not totipotent or pluripotent
    • i.e adult stem cells or somatic stem cells
    • heimatopoietic stem cells
  4. Stem cells have 3 characteristics
    • undifferentiated cells
    • capable of self renewal via mitotic cell division while maintaining undifferentiated state
    • able to differentiate into specialised cell types under presence of appropriate chemical signals
  5. Gene therapy
    • is a technique for correcting defective gene responsible for disease development
    • Introducing a copy of a normal functional gene into target cells with non-functional genes
    • A vector is required - can be viral/non-viral.
    • Newly introduced gene is transcribed and translated for synthesis of normal functional protein product thus restoring correct function of target cells, altering phenotype
  6. Types of gene therapy vectors
    • Retrovirus
    • Adenovirus
    • Adeno-Associated virus
    • Lipsomes
  7. 2 types of gene delivery methods
    • Ex Vivo (outside body)
    • In Vivo (inside body)
  8. Ex Vivo
    • cells are removed from the body and cultured in vitro
    • gene of interest is inserted
    • these transformed cells are then allowed to multiply to a sufficient number and then return to the body by infusion or transplant
    • patient's own cell used, no rejection
  9. In Vivo
    • gene of interest is introduced directly into cells with the body
    • gene could be introduced either systematically or topically
    • not as controlled or specific compared to ex vivo
  10. Severe combined immunodeficiency (SCID)
    • inheritable congenital disease
    • poor or no immune response
  11. X-linked SCID
    • mutated interleukin-2 receptor gamma (IL2RG) gene
    • sex-linked recessive - IL2RG gene is found on X chromosome - hence more affected males XY (X dominant over Y)
  12. IL2RG
    • controls production of the common gamma chain subunit of the protein receptor on the surface of immature blood stem cells in the bone marrow.
    • The receptor directs the development of T, B and natural killer cells of the immune system from blood stem cells.
  13. Mutations to the IL2RG gene
    • results in production of a non functional version of the common gamma chain or no protein at all.
    • Without this common gamma chain, important chemical signals are not relayed to the nucleus. - near complete failure of immune system due to immature T B and natural killer cells.
  14. ADA-SCID
    adenosine deaminase deficiency
    Autosomal recessive disorder of the ADA gene at chromosome 20
  15. ADA (adenosine deaminase) is an enzyme
    • involved in the breakdown of dATP (deoxy-adenosine triphosphate)
    • Lack of ADA results in dATP accumulation.
    • Immature T and B cells are affected by the toxic effects of dATP accumulation and hence fail to reach maturity.
  16. Gene therapy of SCID using retroviral gene delivery
    • Normal gene is cloned and inserted into an inactivated, non pathogenic retrovirus
    • Target cells are then isolated from patient and cultured in the lab
    • Target cells are then infected with the retroviral vector and further cultured before they are re-introduced into the patient
  17. Advantages of using stem cells in gene therapy
    • capable of differentiating into different cell types
    • capable of continual mitotic divison -> ensures that inserted gene is not lost
  18. Disadvantages
    • Injections with retroviral T cells had to be repeated as T cells live for only 6-12 months in the blood.
    • Risk of insertional mutagenesis (inapporopriate insertion of retroviral vector near a proto-oncogene promoter) -> Uncontrolled proliferation of T cells
    • Risk of stimulating an immune response
    • Risk of insertional mutagenesis
  19. Cystic Fibrosis (CF)
    • autosomal recessive trait affecting chromosome number 7
    • caused by inheriting two mutated copies of gene coding for a transmembrane protein (cystic fibrosis transmembrane conductance regulator) (CFTR)
  20. NORMAL CFTR protein
    is a transmembrane chloride channel that allows the efflux of chloride ions out of epithelial cells
  21. Mutation in CF
    • involves the deletion of 3 base pairs (equals to 1 codon) from the CFTR gene.
    • As such, mutated CFTR protein is missing the A.A phenylalanine at position 508.
  22. 3D configuration of mutated CFTR protein changed
    • Preventing normal efflux of Cl- ions out of epithelial cells.
    • This build up of Cl- ions in the cells, attract water into the epithelial cells and away from the external mucus layer.
    • Thick and sticky mucus forms and clogs up the airways of the lungs, pancreatic duct, bile duct, sweat gland ducts and reproductive ducts.
    • Bacteria thrive in the thick mucus causing infections. i.e pneumonia, diabetes (blocked insulin release)
  23. Liposomes
    are phospholipid vesicles that encapsulate the gene of interest, and fuses with the plasma membrane of the target cell.
  24. Gene therapy of CF using non-viral gene delivery
    • Normal CFTR gene is cloned into a plasmid, which is placed into cationic liposomes and sprayed into the nose and mouth of CF patients as an aerosal spray.
    • Cationic to minimise repulsion btw liposome and cell membrane as both are -vely charged.
    • Cationic liposomes fuse with the cell membrane of tracheal cells, releasing the normal CF gene into the cytoplasm of cells.
  25. Gene therapy of CF using non-viral gene delivery
    • This introduced CF gene then enters the nucleus, transcribed into mRNA and translated into the normal CFTR protein.
    • Normal CFTR protein embeds into cell membrane and begins to transport Cl- ions out of cells, thinning the mucus and water moves out of cell to dilute thick sticky mucus and alleivate CF symptoms.
  26. Disadvantage of CF gene therapy
    • multiple reapplications are required, since the DNA delivered via liposomes does not integrate into the chromosomes & epithelial cells constantly being shed, thus delivered gene is lost
    • Low efficiency of transfer due to non-specificity, gene does not incorporate into target cells genome
  27. Advantage of CF gene therapy
    Liposomes can be safely administered to patients without invoking any immune response
  28. Factors that prevent gene therapy from becoming an effective treatment for genetic diseases
    • Difficult to get DNA to integrate into target cell genome
    • Short-lived nature of gene therapy
    • Difficulty in controlling gene expression
    • Risk of stimulating an immue response Virus vector may develop virulence
    • Incorrect gene insertion (insertional mutagenesis) may cause cancer
    • Size constraint (difficult to find suitable vectors for large genes)
    • Difficulty in treating multi-gene disorders (impossible to introduce many genes into target cell at the same time)
    • Difficulty correcting a disease which is due to presence of a dominant allele (as dominant allele has to be removed first, before introducing copies of the recessive normal allele)
    • Not applicable to non dividing cells
    • Low efficiency of liposomes
  29. Social Considerations for Gene therapy
    • Directed to treatment of diseases with an enomous social impact
    • Difficulty in determining which conditions are normal and which are disorders
    • Gene therapy very expensive -> accessible only to wealthy, possible genetic enhancements creating an advantage for those who can afford treatment
    • Concerns about widespread use of gene therapy making society less accepting of people who have genetic diseases or less abled
  30. Ethical Considerations
    • Germ-line gene therapy (correct genetic defect in future generations, forever changes genetic makeup of an individual's descendants)
    • Safety of gene therapy to humans(unknown risks)
    • Protection of privacy and confidentiality of medical information
    • Concerns that gene therapy maybe abused for genetic enhancement -> eugenics
  31. Plant Cloning (Micropropagation/Plant Tissue Culture)
    asexually propagate plants to produce genetically identical descendents
  32. Steps for micropropagation
    • Performed under aseptic conditions to prevent bacterial and fungal contamination
    • An explant is taken from a stock plant that possess desirable characteristics -> meristematic tissue (active mitosis occurring and totipotent state, disease-free)
    • Explants sterilised with bleach/sodium hypochlorite
    • explant cells cultured in growth meidium (plant growth regulators auxin & cytokinin, sucrose, A.A, vitamins and organic ions)
    • cells divide by mitosis to form callus(mass of totipotent actively dividing cells)
    • callus maybe subcultured onto separate culture vessels
    • using identified ratio of auxin & gibberellin (plant growth regulators), callus differentiate into plantlet
    • plantlet transffered into soil, allowed to grow under controlled conditions in greenhouse
  33. Advantages of plant tissue culture
    • Mass production of many copies of genetically identical plants with desirable traits is possible
    • Less space required
    • Easier to transport
    • Disease free plants produced
    • Genetic Modification of plant cells is possible during tissue culture
    • Not affected by environmental conditions
    • Plants difficult to germinate from seeds easily produced via tissue culture
  34. Disadvantages of plant tissue culture
    • Expensive as labour cost of more than 70%
    • Infected stock plant produce many infected progeny -> some very hard to disinfect
    • Growth medium for plant species not known, cannot be successfully tissue cultured
    • Genetically identical plants are susceptible to new diseases/pests
    • Undergo genetic changes to yield progenies that are undesirable
  35. Genetically Modified Organism
    is an organism whose genetic material has been altered using genetic engineering techniques.
  36. Transgenic
    refers to organisms which have inserted DNA that originated from a different species.
  37. GMOs are
    • Faster than conventional breeding
    • A gene for desirable trait can be identified and cloned
    • All beneficial characteristics of existing variety can be kept and just desired new gene can be added
  38. Bt Corn
    • Genetically altered to express the bacterial Bt toxin (poisonous to insect pest such as European Corn Borer)
    • Transgenic Bt corn produces Bt toxin as a natural insecticide
    • Significance: Yield increase as Bt corn less susceptible to attacks by corn borers
    • Use of environmentally harmful pesticides reduced. Use of DDT can be stopped.
  39. Golden Rice
    • Designed to produced beta-carotene - precursor of Vitamin A
    • Created by transferring 2 beta-carotene biosynthesis gene into the rice genome : psy gene and crt gene
    • Significance:
    • nutritional value improved, can be used as Vit A supplement
    • complement existing efforts to reduce blindness and other VAD induced diseases in impoverish countries
    • engineered Atlantic salmon contains growth hormone genes that stimulate the fish to grow to market size in about half the time it takes for normal salmon
    • Inserting 2 foreign genes - 1st gene is growth hormone gene from Chinook salmon 2nd gene is a promoter that activates 1st gene taken from ocean pout
    • The pout promoter gene is able to disrupt the salmon's normal cycle of producing growth hormones only during the summer months.
    • Significance:
    • Faster growth rate
    • Make fish farming more environmentally sustainable (supply could be increased)
  41. Social Implications
    • Higher yield and quality of products due to:
    • Improved nutritional value
    • Increased resistance to disease
    • Increase tolerance to environment

    Mass production of pharmaceuticals
  42. Social Implications
    • Threat to human safety:
    • Use of vectors which confer antibiotic resistance
    • These genes also enter transformed cells along with desired gene
    • Transgenic food eaten, gene may pass to E.Coli or to other potentially harmful bacteria in the environment
  43. Social/ethical Implications
    • Unknown effects of GM products on human health and disease:
    • Development of unknown tocix or allergenic components
  44. Social Implications
    • Threat to safety of environment:
    • Seeds from GM crops carried to somewhere else and may establish themselves as weeds as they can be resistance to herbicide
  45. Social Implications
    Cross pollination may occur btwn genetically engineered crops and their wild relative, spreading resistance to weeds forming superweeds
  46. Social Implications
    • GMOs might upset ecological balance:
    • Affect the balance of the food chain/web in the enviroment or ecosystem
  47. Social Implications
    GMOs lead to reduction in biodiversity as they are better able to withstand enviromental stress, reduce genetic diversity and variation in gene pool
  48. Social/ethical Implications
    • Monopolistic behaviour of biotechnology companies :
    • Practise terminator seed technology - causing 2nd generation seeds to be sterile - world food production hence dominated by a few large biotechnology companies
    • increasing dependence on such companies
    • rich countries hence can exploit the poor
  49. Ethical Implications
    • Infringing nature and applying GMO to humans :
    • GMO tampering with nature
    • GM of animals draws parallel that human GM for enhancement may one day occur
  50. Ethical Implications
    • Violation of animal rights:
    • Animals exploited for food - animals might not be biologically capable of withstanding additional stress of increase production of meat etc
    • Animals exploited for medical research
  51. Ethical Implications
    • Religious concerns/dietary restrictions:
    • use of animal genes in plants change fundamental vege nature of plants
    • religious beliefs maybe infringed with transfer of certain animal genes
  52. Ethical Implications
    • rights of patenting GMOs
    • patent animals is unethical as it reduces them to level of objects
  53. Ethical Implications
    Labelling issues - labelling to indicate that it is GMO but biotech company not willing - consumers unknowingly consume (harmful to them or dietary restrictions (contains allergens) or religious)