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Biology Application Syllabus: Applications of Molecular and Cellular Biology
Applications of Molecular and Cell Biology
1.
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Explain the unique features of stem cells.
Unspecialized
Ability to undergo extensive proliferation and self-renewal, with daughter cells possessing same developmental and
replicative potential as parent cell.
Ability to differentiate to produce specialized cells
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Embryonic Stem Cells
Derived from blastocyst, a ball of cells that develop from zygote after 4-5 days of fertilization
Blastocyst consists of inner cell mass, trophoblast and blastocyst cavity
Inner cell mass are pluripotent
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Adult Stem Cells
Descendents of pluripotent stem cells (multipotent)
Found in various tissues – e.g. bone marrow.
Replaces cells that are lost due to tissue damage or injury
2.
Explain the normal functions of stem cells in a living organism, using appropriate examples to illustrate.
In Brain
CNS neural stem cells give rise to both neurons and glial cells
Implications: In the case of Parkinson Disease – a degenerative disorder of CNS that impairs one’s motor skills and
speech due to insufficient formation and action of dopamine, produced by dopaminergic neurons of the brain
In Bone Marrow
Blood contains different types of cells like red blood cells and various types of white blood cells and platelets.
Hematopoietic stem cells gives rise to lymphoid and myeloid progenitor cells which are precursors to specialized cells
Lymphoid progenitors specialize into B cell, T cell and Natural Killer cells
Myeloid progenitors specialize into Neutrophil, Basophil, Eosinophil, Monocyte, Platelet producer cells and Red Blood
cells
In Epidermis
Epidermal stem cells will produce new cells (keratinocytes) which migrate to the surface of the skin to replace damaged
or shed epidermis cells
In Epithelium of Intestine
There are times when lining of gut is digested and have to be replaced by new cells produced by the intestinal stem
cells.
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
3.
Describe two types of genetic diseases e.g. SCID (severe combined immunodeficiency) and cystic fibrosis, using viral
and non viral gene delivery systems, that can be treated with gene therapy.
Genetic Therapy is the addition of normal (functioning) genetic material such that a genetic defect or disease can be
corrected, by usually introducing a healthy, normal allele.
Gene therapy can potentially treat disorders where normal treatment has failed, and provide a permanent solution to
genetic diseases if germ-line cells are “corrected”. Somatic cell gene therapy would be effective for the patient’s lifetime.
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Severe combined immunodeficiency
Severe form of heritable immunodeficiency
These disorders may lead to reduced/malfunctioning T- and B-lymphocytes, responsible in fighting infection.
X-linked SCID – Caused by mutations in gene encoding the common gamma chain, a protein shared by receptors for
some interleukins, which are involved in the proper development and differentiation of T and B cells.
Non-functional common gamma chain will lead to a near complete failure in the immune system. The
common gamma chain is encoded by gene located on X-chromosome, thus it is inherited in an X-link
recessive pattern.
Adenosine deaminase – Caused by defective enzyme, adenosine deaminase (ADA), which is involved in breaking down
of purines. This is an autosomal recessive disorder. Disorder will cause deoxyATP to
accumulate, which at high concentrations, is toxic to T and B cells, leading to their apoptosis.
Treatment
 Best hope for X-linked SCID is Stem cell transplant
 Stem cells introduced into the body in hopes that the new cells will rebuild the immune system.
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In the case of malfunctional ADA, possible treatment includes enzyme replacement therapy. Weekly injection of
ADA prevents buildup of deoxyATP. It is a lifetime treatment.
By viral-delivery method. Involves removing cells from infected and inserting healthy genes into these cells using
retrovirus, and transplanting them back.
Production of ADA is temporary, so regular infusions of recombinant T lymphocytes needed.
Cystic Fibrosis
 Commonly afflicts Caucasians
 Autosomal recessive gene on chromosome 7
 Deletion mutation resulting in loss of phenylalanine in the protein
 Cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein is either missing or defective
 CFTR controls movement of Cl- into and out of cells.
 In lungs, defective CFTR leads to Cl- not transported out of epithelial cells. Na+ is retained due to charge balance,
decreasing the water potential in the cells, resulting in water retained. Undiluted mucus hence is viscous.
 Congestion of the mucus may lead to lung infection, severe breathing difficulty
 Pancreatic duct choked by thick mucus would prevent release of enzymes, causing indigestion.
 Thick mucus layer in intestines reduces absorption of digested food
 Sweat glands produce salty sweat, since defective CFTR results in NaCl not being reabsorbed. (Forms basis of
diagnosis)
Treatment
Non-viral delivery system – Liposomes
 Somatic cell gene therapy
 Insert functional CFTR gene into plasmid vector packaged in microscopic lipid envelopes (forming liposomes)
 Deliver by aerosol spray
 Gene may enter nucleus, integrate into genome and produce normal CFTR protein
 Temporary treatment as epithelial cells are constantly shed
Viral delivery system – Adenovirus
 Virus modified to remove genes that cause infections and replicate, and to allow accommodation of CFTR gene
 Modified virus used as vector, can effectively infect cells and transfer genetic material into cell
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
4.
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Explain the factors that keep gene therapy from becoming an effective treatment for genetic diseases.
Short-lived nature of gene therapy, due to problems with integrating therapeutic DNA into genome
Rapidly dividing nature of many cells prevent gene therapy from achieving long-term benefits, need for many rounds
of therapy
Risk of immune response, reducing effectiveness
Viral vectors may cause immune and inflammatory responses towards these vectors.
Viral vectors may resume ability to be pathogenic
Inadequate understanding of the interactions of vector and host.
Ineffective in treating multi-gene disorder caused by combined effects of variation in many genes.
May result in tumor, because of insertional mutagenesis.
5.
Discuss the social and ethical considerations for the use of gene therapy.
For Somatic cell gene therapy
Potential in treating desperately ill patients, where
conventional treatment has failed. Civic, religious,
scientific and medical groups have agreed in principle that
somatic-cell gene therapy is appropriate for humans
Somatic gene therapy corrects defects present in
individual alone, without impacting the genetic
information of successive generations
For Germ-line gene therapy
True cure, not simply symptomatic
Only effective way of addressing some genetic diseases
Expense and risk of somatic-cell therapy for successive
generations avoided
Medicine should respond to prospective parents at risk of passing
on serious genetic diseases
The community has the right to free inquiry
Even though the research would place embryos at risk, once
successful techniques are developed, the therapy would help
parents avoid the moral dilemma of disposing “defective”
embryos.
Against Gene therapy
Involves alteration to the genes of germ-line cells affecting successive generations
Potential of abuse to the technology
Difficulty in conducting long-term clinical research
Gene therapy candidates are usually children, who are too young to understand the ramifications of gene therapy
Conflict of personal and interests of society in caring for children with serious genetic defects
Justification of resource allocation. High cost accrued by the research
Argument specific to Germ-line Gene Therapy
Long term effects of germ-line gene therapy holds too much uncertainty and risk
May open door to attempts at altering human traits not associated with disease, exacerbate problems of social discrimination.
Eugenics.
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
6. Discuss cloning in plants.
Clone: Cell(s) or organism that is descended from a genetically identical ancestor (e.g. bacterial colony)
Organism descended asexually from a single ancestor
Vegetative reproduction: Asexual reproduction by plants in which new daughter plants are growth without seeds or spores
E.g. of natural vegetative propagation: Rhizomes (Ginger), runners (Strawberry), suckering (banana)
Horticulture: Man-made methods of vegetative reproduction using simple cloning methods such as cuttings, grafting, layering
Tissue Culture & Micropropagation
Tissue Culture:
Propagation of plants through placement of small amounts of undifferentiated tissue/single cells in
artificial environment
Micropropagation: Plant tissue culture that is used to clonally produce thousands of plantlets
(i.e. vegetative reproduction on a large scale)
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Technique of plant tissue culture
 Preparation of Explant
Explant: Any portion of plant tissue that is taken to initiate a tissue culture, from which the whole plant can be
regenerated.
Examples of ideal plant tissues: flower buds, leaves, meristem
Donor plants should carry desirable traits, higher yield, better quality of fruits/flower, gene resistance
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Obtaining Aseptic Cultures
 Explant surface-sterilized with sodium hypochlorite (Clorox™)
 Cut into small fragments
 Explant transferred to culture medium containing mineral nutrients, vitamins, carbohydrate sources and plant
growth regulators
 Medium thickened with agar to create gel that supports the explants during growth
 Explant are pressed into surface of agar to ensure good contact (support) with medium
 Incubate for 1-9 weeks
 Sterility of containers and work bench should be maintained at all times
 Callus, a mass of undifferentiated tissue would develop
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Rapid Multiplication of Callus
 Calli repeated subcultured onto new culture media to obtain required multiplication rates
 Proportion of cytokinin:auxins controlled
 Higher proportion of auxins is required for callus proliferation (auxin:cytokinin intermediate)
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Establishment of Plantlet
 Plant tissue induced to form plantlets by culture media modifications
 Adventitious shoots needs high cytokinin:auxin ratio
 Lowering cytokinins or eliminating totally, and addition of auxin promote root development
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Sugar levels ↓, light intensity ↑, providing suitable conditions to stimulate photosynthesis, and other changes
required for autotrophic growth.
Acclimatization
 New plantlets are acclimatized by slowly weaning them from a high humidity, low light, and warm
environment to a normal growth environment
 Plantlet taken from the culture, washed off of agar, soaked in fungicide, grow in sterile soil
 Plantlet grown in greenhouse for 4-8 weeks
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
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Advantages of Micropropagation
 High fecundity – potential reproductive capability
 Genetic uniformity – All possess the desirable features
 Produces disease-free plants
 Produces rooted plantlets ready for growth
 Often produces more robust plants
 Multiplying plants which produce seeds in uneconomical amounts (e.g. sterile bananas)
 Independent of climatic changes – Tissue culture can be carried out in controlled environment
 Takes up little space
 Large quantities of plantlet can be transported easily
 Important tool for genetic engineering - only viable method of regenerating modified cells (e.g. cells in protoplast
fusion)
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Disadvantages of Micropropagation
 Expensive
 Infected plant sample can produce infected progeny
 Losses can incur during transfer from in vitro to acclimatization
 Higher than acceptable levels of somatic variation.
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Somatic embryogenesis
 Plant cells stimulated to form embryos in plant tissue culture; embryos are called somatic embryos
 Culture tissue culture to form callus.
 Plant growth regulators manipulated to induce callus formation and subsequently changed to induce embryos
 ↑auxin and ↓nitrogen to induce embryo formation
 Somatic embryoids can be packaged as functional seeds in capsule (protective, hydrated gel with nutrients .etc)
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Protoplast cultures
Protoplasts are plant cells without their cell walls
Reasons for protoplast culture
 No cell wall – can introduce genetic material into cell with more ease
 Selected DNA can be introduced into the cell  transformed protoplasts  transformed plants
 Two or more protoplast can be fused together to form somatic hybrid (Protoplast fusion)
Allows breeders to overcome reproductive barriers and combine good traits of two plants
Generating Polyploids
 Banana – Sterile triploid – seedless. Doubling chromosome number will allow pairing of homologous chromosomes
in meiosis, allowing it to be fertile
 Polyploidy increase vigour in plants (e.g. Octoploid blackberries/strawberries are larger)
Note: Fusion of protoplasts of two different varieties form heterokaryons
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
Procedures for protoplast culture
 Choosing explant
 Meristematic cells used.
 Surface sterilization of tissue with sodium hypochlorite
 Isolating Protoplasts
 Using leaf explants, lower epidermis peeled
 Cells plasmolysed in mannitol, sorbitol or sucrose to free cytoplasm from cell wall and break plasmodesma
connections
 Mechanical disruption / enzymatic treatment (fungal pectinases, hemicellulases, cellulases) to remove cell
walls
 Protoplast suspension collected by centrifugation, washed and separated from cell debris by flotation on
sucrose cushion.
 Protoplast Culture
 Protoplast can be induced to fuse by chemicals such as Calcium or polyethylene glycol (PEG), makes
protoplasts ‘sticky’
 Protoplast can be induced to fuse by Electrofusion, exposing cells to small amounts of currents
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Anther culture
 Pollen grains are used for explant
 Callus Culture (Refer to above)
 Produces haploid anther calli
 Colchicine can be used to disrupt formation of mitotic spindle (prevents separation of chromatids at
anaphase), resulting in homozygous diploid plants, restoring fertility
 Mutation can be induced in anther culture to select for desirable mutants.
 Phenotypic effects of all genes can be seen (no masking), thus lethal genes are eliminated because such
recessive genes are expressed
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
7.
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Explain the significance of genetic engineering in improving the quality and yield of crop plants and animals in solving
the demand for food in the world.
Significance
 Genetic engineering offers chance to add new genes directly without relying on sexual reproduction
 Offers possibility of ‘designer’ plants and animals with desirable properties
 Selective breeding is very slow and can take many years to establish desired phenotypic characteristics for animals
with long gestation period. Recombinant DNA technology greatly increases speed and range of selective breeding.
Genetic engineering refers to the manipulation of genetic material. This includes altering the DNA of an organism to
suppress or enhance the activities of its own genes and combining genetic material of different species. Universal
genetic code allows organisms to carry genes from unrelated organisms that have been artificially introduced.
Transgenic organisms or genetically modified organisms are organisms with their genes altered.
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Ways in which genetic engineering can be used to improve quality and yield of animal in solving the demand for food
Improvement in meat production
 Pigs
 Transgenic pigs with meat rich in omega-3 (fat-1 gene from Caenorhabditis elegans)
 Porcine somatotropin for leaner meat – Porcine somatotropin (PST) increases feed efficiency in pigs by 15-20%,
reduces fat deposition in pigs.
Improvement in milk yields and milk quality
 Bovine somatotropin for increase in milk yield (bovine – cows)
 Use of lactating animals to produce novel secretion of human proteins in their milk, which can be extracted and
used pharmaceutically
 Insertion of gene for factor IX (blood-clotting factor) into sheep, produced in milk of transgenic animals
Disease-resistant livestock
 Produce livestock resistant to diseases (very limited no. of genes are currently known to confer resistance to
diseases)
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Biology Application Syllabus: Applications of Molecular and Cellular Biology
8.
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Discuss the ethical and social implications of genetically modified organisms.
Benefits
 Farmer
 GM Crops result in higher yield.
 Less usage of pesticides – Savings in pesticides, labour costs
 Consumer
 Cleaner food, fewer pests and chemical contamination
 Food with improved nutritional value
 Food with better storage properties and taste
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Ethical and social implications (Positive)
 Increased disease resistance
More rapid and precise inclusion of genes via genetic engineering, such that coupled with proper management,
might be possible to reduce buildup of resistance in pathogen, though they would eventually be resistant due to
natural selection pressures, genetic engineering allows faster response to new pathogen “strains”
 Increased tolerance to unfavourable conditions
Genes could also be introduced into crops that would help plants tolerate less favourable condition, which would
greatly increase the proportion of arable land.
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Ethical and social implications (Negative)
 Pest Resistance
Occurrence in response to extensive application of herbicides (e.g. continued exposure of pests to Bt plants will
inevitably lead to resistance)
 Effect on Non Target Organisms
Unforeseen effects of the toxin on organisms that are not pests of the crop
 Gene Flow and Superweeds
Transgenic plants may pass their new genes to close relatives in the wild. This spontaneous hybridization may lead
to “superweed”.
 Biodiversity
GM crops may outgrow local native species in the environment. If they are capable to compete with the wild, when
accidentally released into the wild, may disrupt natural ecosystem
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