1.4 Aspects of Genetic Engineering

• What are the 6 steps in gene technology

  1.- the gene is identified and is either cut out of the chromosomes, made by reverse transcription of mRNA or synthesised directly in the laboratory.

  2.- Copies of the gene are made by polymerase chain reaction

  3.-3 The gene is inserted into a vector

  - an organism

  or structure that is able to deliver the gene into

  the required cells.

  4 The vector inserts the gene into the cells.

  5 The cells that have been successfully

  transformed are identified and cloned.

  6 The cloned cells are used in industrial or

  medical situations.

  The steps involved in this procedure are described

  here with particular reference to the production

  of recombinant bacteria that make insulin.
• What is a recombinant organism

  any organism that contains and expresses genes that originated in another organism
• Whay is a genome


  this is a complete set of genetic material of an organism
• What is recombinant DNA

  DNA produced by combining DNA from different sources
• What is genetic engineering?


  The direct manipulation of genes for practical purposes.;altering the genetic material/genome of an organism
• What is DNA?

  deoxyribonucleic acid; genetic material that codes for different proteins in our body
• TRIAL IN THE USA

  - they introduced the gene into normally harmless adenoviruses and uses these to carry gene into passages of gas exchange system

  
  

  Result: gene did enter some of the cells but some of the volunteers experienced unpleasant side effects as a result of infection by the virus
• TRIAL IN UK

  Trials began in 1993

  
  

  -normal allele was inserted into liposomes which were then sprayed as an aerosol into the noses of nine volunteers

  - the hope was that the liposomes would be able to move through the lipid layers in the plasma membranes of the cells lining the respiratory passages carrying the gene with them

  
  

  Result: trial succeeded in introducing the gene into a few cells lining noses of volunteers but effect only lasted for a week because these cells have only a very short lifespan and are continually replaced.
• Who is a good candidate for gene therapy?

  Someone with with one faulty allele and one normal allele is able to make enough CFTR protein to remain healthy

  
  

  We don't need to remove the genes that are already there, we just need to get a correct, dominant allele into cell and in theory be able to make enough of CFTR protein to allow the cell to work properly
• Implications of cystic fibrosis?

  It is a genetic disease in which abnormally thick mucus is produced in the lungs and other parts of body

  
  

  - person very prone to bacterial infections in lungs as mucus is difficult to remove and bacteria can breed in it

  
  

  - the mucus also can block ducts like the pancreatic duct

  
  

  -90% of men with this are sterile because thick secretions block ducts in reproductive system
• This results in a relatively high conc of chloride ions outside the cell. This reduces the water potential below that of the cytoplasm of the cells so water moves out of the cells by osmosis, mixes with mucus there, making it thin enough for easy removal by sweeping movements of cilia

  
  

  For someone with cystic fibrosis much less water moves out of cells so mucus is thick and sticky, the cilia or coughing cannot remove it all
• What is the importance of this protein?

  This protein sits in the plasma membrane and allows chloride ions to pass out of cells

  
  

  The recessive alleles (there are several different faulty ones) code for an incomplete or faulty version of this protein which does not act properly as a chloride ion transporter

  
  

  In a healthy person:

  The cells lining the airways and in the lungs pump out chloride ions through the channel in the membrane formed by CFTR
• The CFTR gene


  The gene that encodes the CFTR protein is found on chromosome 7

  - the most common defective allele is the result of the deletion of three bases

  - the CFTR protein made using this allele is therefore missing one amino acid

  -the machinery in the cell recognizes that this is not the right protein and does not place it in the plasma membrane
• Attempts have been made to treat...

  1) cystic fibrosis

  2) SCID (severe combined immunodeficiency disease)
• What is germ line gene therapy?

  This involves changing the genes in cells that would go on to form gametes and therefore possible zygotes. If this were done, then all the cells in the new organism would carry the genetic modification
• What is somatic gene therapy?


  This is when the cells being genetically modified are body cells. They are not involved in reproduction

  
  

  (If the genes in them are modified, the effect stops there, in that person and will not be passed on to any of the person's offspring)
• What are the two main categories of gene therapy?

  1) somatic gene therapy

  2) germ line gene therapy
• Tomatoes

  - enzyme POLYGALACTURONASE breaks down cell walls during tomato ripening

  - if this enzyme is inhibited, tomatoes can be harvested when partially ripe, transported to market in a firm state then ripened for sale

  - the bacterium Agrobacterium tumifaciens can transfer part of its Ti- plasmid (T-DNA) into host plant genome
• What kind of conditions has it been used for?

  -Cancer

  -Infectious diseases

  - degenerative disorders

  
  

  NB: GMOs include those that don't include recombinant technology but also gene therapy (other methods of manipulating genome)
• What is gene therapy?

  Gene therapy is an application of genetic engineering.It involves the transfer of a normal functioning gene into a person who has a genetic disorder. There are a variety of methods of delivery of genes into cells using:

  
  

  1) viruses that are taken up by specific cells

  2)liposomes (small phospholipid bound spheres)

  3) plasmids injected directly into cells
• 3.Insulin


  Bacteria Escherichia coli is used to produce insulin for pharmaceutical use

  
  

  As the bacteria grow and multiply, insulin is collected and purified

  
  

  Insulin collected from bacteria is in the form of proinsulin which must be cleaved with enzymes to give human insulin
• 2. Erythropoietin (EPO):


  What is it: it is a protein produced in the kidneys

  
  

  What does it do?

  It stimulates the stem cells in bone marrow to make RBCs

  
  

  - persons who suffer from kidney failure and require kidney dialysis can become anaemic

  - this is because dialysis removes waste and toxins from body but also removes erythropoietin from blood so insufficient new RBCs are made

  -recombinant DNA technology was useful in isolating gene coding for erythropoietin and using bacteria to manufacture protein

  -erythropoietin is a valuable medicine for reducing anaemia in dialysis patients
• 1) Tissue plasminogen activator

  -when wounds start to bleed, a bleed clot forms to stop the blood flow and later the blood clot is dissolved

  
  

  - heart attacks and strokes are caused by blood clots that form in the blood vessels that transport blood to heart and brain

  
  

  - the blood of mammals including humans contains an enzyme called plasmin that dissolves clotting proteins

  
  

  - plasmin is found in the blood in the inactive form called plasminogen

  
  

  - another enzyme called tissue plasminogen activator activates the conversion of plasminogen to plasmin

  
  

  TPA is a medicine used for treating patients at risk from strokes and heart attacks (recombinant dna technology was useful in isolating gene that codes for TPA in humans and then using this gene along with vectors in bacteria to manufacture protein in large amounts for medicinal purposes)
• Recombinant DNA applications in medicine

  1) Tissue plasminogen activator

  2) Erythropoietin

  3) Insulin
• Example

  If it were necessary to search through the entire human genome for insulin gene, it would be costly and time consuming. The alternative is to use mRNA from cells in pancreas that normally produce insulin
• Why waste time with cloning, why not just get nucleic acid probe and detect the gene of interest in DNA isolated directly from organism?

  Most protein-coding genes are present at one part per million or less therefore finding it is difficult and then we would still need many copies for experiment
• How can we detect the gene of interest?

  By using a nucleic acid probe that has the nucleotide sequence that is complementary to the gene of interest

  
  

  We know how it make the nucleic acid probe because we may have info on the protein sequence and this gives info on its DNA sequence or we may have info from closely related species from scientific research
• What is a genomic library?

  A genomic library is the complete set of bacterial clones carrying copies of recombinant DNA from the organism that has the GOI
• What is antitrypsin?


  Antitrypsin inhibits the protease trypsin which is released by phagocytes in the lungs during infections.
• What is antithrombin?

  Antithrombin inhibits thrombin, an enzyme that promotes blood clotting . Antithrombin works to thin the blood slightly so that it doesn't clot too much
• Continued

  4) foreign genes can "pollute" non-GM and organic crops which require certification that they provide 'GM- free' foods

  
  

  5) GMO crops require more herbicide applications and just as much pesticide as non GM crops so there is no advantage in terms of cost to farmers or reduction in chemicals used in agriculture

  
  

  6) Farmers cannot keep seed for sowing for the following crops as GM crops do not 'breed true' this favors large-scale commercial farmers and not many farmers in developing countries
• Moral and ethical issues of GMOs

  1) antibiotic resistance genes used to identify GMOs could 'escape' and be transferred to pathogenic organisms making some antibiotics redundant

  
  

  2) Herbicide resistance genes could be transferred in pollen to weed species and can lead to development of 'super weeds' that are resistant for herbicides

  
  

  3) Foreign genes could be transferred to wild relatives of our crop plants so changing their genomes, this may 'pollute' those species that may prove useful sources of genes for crop development in future
• Some important points to note

  -using these organisms is the large production and therefore cheaper prices of the substances concerned

  
  

  - transgenic microorganisms do not compete well in the natural environment as they are engineered to produce substances that give them no advantage

  
  

  -lethal genes are added to the microorganisms so that they die when removed from the conditions of the culture
• GMOs in medicine

  1) insulin production

  2) human growth hormone

  3) thyroid stimulating hormone

  4) factor VIII- a blood clotting protein

  5) vaccines eg: for influenza

  6)many chemicals for research including monoclonal antibodies

  7) Human antithrombin to treat blood loss produced in transgenic goats

  8) Human alpha- anti trypsin to treat emphysema produced by transgenic sheep
• 4) dietary supplements

  Example: bacteria

  
  

  Feature that has been added: tryptophan (amino acid)
• 3) Production of human proteins for medical use

  Example: yeast

  
  

  Feature that has been added: insulin, HGH, vaccines
• 2) wine production

  Example: yeast

  
  

  Feature that has been added: improving taste and colour stability of wine as well as avoid production of undesirable compounds (histamines)
• 1) Production of animal protein

  Example: bacteria

  
  

  Feature that has been added

  
  

  - chymosin (rennin) for cheese making

  - somatotrophin for injecting into cattle to improve milk production
• Features of GM microorganisms?

  1) Production of animal protein

  2) Wine production

  3) Production of human proteins for medical use

  4) dietary supplements
• 4) Production of human proteins in milk

  Example: dairy cattle

  
  

  Feature that has been added: genes for human lysozyme and other proteins found in human milk