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  • This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981) which are the most difficult of all cells to genetically manipulate. The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The DNA construct (usually about 100 to 200 copies in 2 pl of buffer) is introduced by microinjection through a fine glass needle into the male pronucleus - the nucleus provided by the sperm before fusion with the nucleus of the egg. The diameter of the egg is 70 µm and that of the glass needle is 0.75 µm; the experimenter performs the manipulations with a binocular microscope at a magnification of 200 x. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.
  • 2. Embryonic stem cell-mediated gene transferThis method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.
  • 2. Embryonic stem cell-mediated gene transferThis method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.
  • 3. Retrovirus-mediated gene transferTo increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.There is also fusion of host cells with membranous vesicles (eg. liposomes) containing DNA.(Plant cells can be modified using, eg. tobacco mosaic virus or the Ti plasmid of Agrobacterium tumefaciens.)
  • Transgenesis

    1. 1. Learning Intentions:to be able to define transgenesisto be able to give explanations about the biological ideas associatedwith how transgenesis worksto be able to discuss the positive and negative implications oftransgenesis
    2. 2. What is transgenesis?Definitions• Transgenesis is the process of introducing an exogenous gene – called a transgene – into a living organism so that the organism will exhibit a new property and transmit that property to its offspring.• A Transgene is the name given to the introduced DNA
    3. 3. Why use transgenesis instead of selective breeding?• More specific — scientists can choose with greater accuracy the trait they want to establish. The number of additional unwanted traits can be kept to a minimum.• Faster — establishing the trait takes only one generation compared with the many generations often needed for traditional selective breeding, where much is left to chance.• More flexible — traits that would otherwise be unavailable in some animals or plants may be achievable using transgenic methods.• Less costly — much of the cost and labour involved in administering feed supplements and chemical treatments to animals and crops could be avoided.
    4. 4. Uses of transgenesis• in toxicology: as responsive test animals (detection of toxicants);• in mammalian developmental genetics;• to introduce human genes into other organisms (particularly human) for the study of disease processes;• in molecular biology, the analysis of the regulation of gene expression;• in the pharmaceutical industry, the production of human pharmaceuticals in farm animals ("pharming"); targeted production of pharmaceutical proteins, drug production and product efficacy testing;• in biotechnology: as producers of specific proteins;• genetically engineered hormones to increase milk yield, meat production; genetic engineering of livestock in agriculture affecting modification of animal physiology and/or anatomy; cloning procedures to reproduce specific blood lines;• to speed up the introduction of existing characters into a strain/breed for improvement and modification;• developing animals specially created for use in xenografting, ie. modify the antigenic make-up of animals so that their tissues and organs can be used in transfusions and transplants.
    5. 5. Examples of transgenic organism Extended shelf-life tomato (Flavr-Savr) Herbicide resistant soybean (Roundup Ready)
    6. 6. Agriculture Transgenics On the Market (USA) Insect resistant cotton – Bt toxin kills the cotton boll worm • transgene = Bt proteinSource: USDA Insect resistant corn – Bt toxin kills the European corn borer • transgene = Bt protein Normal Transgenic
    7. 7. Biotech chymosin; the enzyme used to curdle milk products • transgene = genetically engineered enzymeSource: Chr. Hansen bST; bovin somatotropin; used to increase milk production • transgene = genetically engineered enzymeSource: Rent Mother Nature
    8. 8. Next Generation of Ag Biotech Products• Golden Rice – increased Vitamin A content (but not without controversy)• Turfgrass – herbicide resistance; slower growing (=reduced mowing)• Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)
    9. 9. Products In The Pipeline• Tomatoes enriched with flavonols • Oranges resistant• Soybean and canola oils with to citrus canker higher levels of vitamin E • Disease-resistant• Vitamin-enriched rice sweet potatoes• Decaffeinated coffee • Pest- and disease-• Bananas to deliver a hepatitis resistant cassava vaccine • Disease-resistant bananas • Potatoes to protect against cholera, E. coli and Norwalk virus • Apples to protect against RSV Benefits of biotechnology – Better food
    10. 10. Tracy (1990-1997): Transgenic Ewe• Genetically modified so that her milk produced a human protein called alpha antitrypsin, a potential treatment for the disease cystic fibrosis.
    11. 11. GTC Biotherapeutics• Pharmaceutical product derived from transgenic goats modified to produce therapeutic proteins in their milk.• The product, ATryn (an antithrombrin) received regulatory approval in the EU in 2006 and in the U.S. in 2008.
    12. 12. Pigs Genetically Engineered To Lack A Sugar-producingGene That Causes Human Bodies To Reject Pig Organs
    13. 13. Biological Processes• First, the desired gene must be extracted from the donor organism.• This is done using restriction enzymes (restriction endonucleases).• It is important that the restriction enzymes cut out the whole gene required.• This gene can then be inserted into host cells (another enzyme, DNA ligase, is very important here)• modules/hormones/en-flash/geneticeng.cfm
    14. 14. Genetic engineering:Recombinant DNA technology
    15. 15. Delivering the DNA into host organism: animals A. Remove eggs B. Fertilize in vitro C. DNA is microinjected into male pronucleus (prior to nuclear fusion) 100-1000 copies of gene D. Implant eggs into surrogate© 2003 John Wiley and Sons Publishers
    16. 16. Number of Number of ova Number ofAnimal species transgenic injected offspring offspring rabbit 1907 218 (11.4%) 28 (1.5%) sheep 1032 73 (7.1%) 1 (0.1%) pig 2035 192 (9.4%) 20 (1.0%)Microinjection is the most common method atpresent and is generally more successful withlaboratory animals than farm animals.The efficiency of microinjection is quite low:Figures in parentheses are percent efficiencycompared to original number of ova injected.(after Hammer et al., 1985)
    17. 17. Embryonic stem cell-mediated gene transfer
    18. 18. Retrovirus-mediated gene transfer
    19. 19. Delivering the DNA into host organism: plants• Two major delivery methods Agrobacterium a biological system based on the plant pathogen Agrobacterium tumefaciens Gene Gun a mechanical method where the DNA is “shot” into plant cells using a gene gun. Tissue culture required to generate transgenic plants
    20. 20. Positive Implications of Transgenesis: Pharming• Many valuable pharmaceutical products can now be made using transgenic animals, a few examples below: – factor VIII blood clotting factor – fibrinogen blood clotting factor – haemoglobin as a blood substitute – human protein C anticoagulant – alpha-1-antitrypsin (AAT) for treatment of AAT deficiency – cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of CF – insulin for diabetes treatment – growth hormones for treatment of deficiencies• These are used to treat human diseases and defects, improving the quality and quantity of life for afflicted individualsexamples taken from:
    21. 21. Positive Implications of Transgenesis: Agriculture Plants• Improving plants• Transgenic methods have now been developed for a number of important crop plants such as rice, cotton, soybean, oilseed rape and a variety of vegetable crops like tomato, potato, cabbage and lettuce. New plant varieties have been produced using bacterial or viral genes that confer tolerance to insect or disease pests and allow plants to tolerate herbicides, making the herbicide more selective in its action against weeds and allowing farmers to use less herbicide.• Transgenic technologies are now being used to modify other important characteristics of plants such as the nutritional value of pasture crops or the oil quality of oilseed plants like linseed or sunflower.
    22. 22. Positive Implications of Transgenesis: Agriculture Animals• Improving Animals• The main aim in using transgenic technology in animal agriculture is to improve livestock by altering their biochemistry, their hormonal balance or their important protein products. Scientists hope to produce animals that are larger and leaner, grow faster and are more efficient at using feed, more productive, or more resistant to disease. Examples of transgenic breeding programs include:• producing faster-growing and leaner pigs that use food more efficiently and resist common diseases• breeding transgenic sheep that grow better wool without needing dietary supplements of sulphur-containing amino acids.
    23. 23. Negative implications of transgenesis GE technology carries certain inherent unpredictability Some facts  Isolation of a gene from its natural environment and integration into entirely different organism  Possible transgenic instability due to triggering of the inbuilt defense mechanisms of the host organism leading to inactivation or silencing of foreign genes.
    24. 24. • Possibilities of integration of foreign gene at a site predisposed to silencing of genes (position effect). – Variance in the levels of expression of the transgene in different environmental conditions (heat, humidity, light…..) – Possibilities of silencing of genes arising in subsequent generations• Biosafety concerns arise from: – Horizontal gene transfer – Genetic contamination – Transfer of allergens and toxins from one life form to another and creation of new toxins and allergenic compounds
    25. 25. ..Biosafety issues in transgenic crops- ConcernsMain  Development of aggressive weeds/ wild relatives by transfer of transgenic traits  Erosion of land races/wild relatives by genetic pollution in centres of origin/ diversity  Harm to the non-target organisms  Development of pest resistance by prolonged use  Monoculture and limitations to farmers’ choice in crop management  Hazard to human and animal health by transfer of toxins and allergens and by creation of new toxins and allergenic compounds
    26. 26. ….GM foods: Allergenicity; ToxicityAllergy It is a hypersensitive reaction initiated by immunologic mechanisms caused by specific substances called allergens.Toxicity New proteins as a result of intended modification Unintended new proteins as a result of the modification Natural constituents beyond their level of normal variation
    27. 27. ….GM foods: nutritional aspects; unintended effects Intended and unintended changes in nutrient levels Bioavailability of nutrients, stability and processing Presence and effect of anti-nutrients Impact of individual changes on overall nutritional profileUnintended effectsRandom integration of transgenes Insertional mutagenesis Disruption of gene functions Production of new proteins Changes in o Phenotype Metabolites o Enzymes Toxins o Genotype
    28. 28. Poorly investigated products, some have beendiscontinued Poor Quality • FlavrSavr tomatoes (Calgene) Negative Consumer Response • Tomato paste (Zeneca) Negative Corporate Response • NewLeaf (Monsanto) Universal Negative Publicity • StarLink corn (Aventis)
    29. 29. • A case study: Golden Rice
    30. 30. The Golden Rice Story• Vitamin A deficiency is a major health problem • Causes blindness • Influences severity of diarrhea, measles• >100 million children suffer from the problem• For many countries, the infrastructure doesn’t existto deliver vitamin pills• Improved vitamin A content in widely consumed cropsan attractive alternativeGolden rice project:
    31. 31. -Carotene Pathway Problem in Plants IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Problem: Phytoene desaturase Rice lacks these enzymes ξ-carotene desaturase Lycopene Lycopene-beta-cyclase Normal Vitamin A -carotene“Deficient” (vitamin A precursor) Rice
    32. 32. The Golden Rice Solution -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Daffodil gene Phytoene synthase Phytoene Vitamin A Phytoene desaturase Pathway Single bacterial gene; is complete performs both functions ξ-carotene desaturaseand functional Lycopene Daffodil gene Lycopene-beta-cyclaseGolden -carotene Rice (vitamin A precursor)
    33. 33. Biological implications1. Ecosystems: the implanted gene could spread to other plants in the environment, this could have unforeseen side effects2. Genetic biodiversity: increases biodiversity as introduces rice plants with novel genes3. Health or survival of individuals: In remote rural areas Golden Rice could constitute a major contribution towards sustainable vitamin A delivery, therefore increase the survival chances of individuals. GR may cause unforeseen health risks, particularly if it is the first GMO to be widely consumed by children.4. Survival of populations: and hence increase survival chance of human populations living in vitamin deficient/poor areas. However, farmers who wish to sell it in markets may not want to take the risks of adopting a new variety (e.g., lower yield, susceptibility to pests and diseases) unless they are compensated with higher prices or yields. However, such higher prices would work against its incorporation into the diets of the poor, possibly causing it to wind up as a niche product for rich consumers.5. Evolution of populations…
    34. 34. • Current breeding and field trialling work is being carried out by the International Rice Research Institute (IRRI) in the Philippines together with PhilRice, the Philippine Rice Research Institute. PhilRice is preparing a submission to the regulatory authority of the Philippines in 2013, which could lead to initial releases to farmers in 2014.