Genetic engineering

• Genetic engineering

  = Manipulation of the genome to achieve a desired outcome.
• Transgenic= organism that a gene from another organisms. Often called a genetically modified organism
• Isolating the desired gene

  = Restriction endonucleases are used to cut the required gene from the DNA of an organism.

  • Many cut the two DNA strands unevenly, leaving one of the DNA fragments a few bases longer than the other strand. Those regions with unpaired, exposed bases are called sticky ends, they make it easier to insert the desired gene into the DNA of another organism and ensure the strands are inserted the right way around.
• Isolating the gene

  • Isolating the mRNA for the desired gene using the enzyme reverse transcriptase to produce a single strand of complementary DNA.
  • Makes it easier to identify the desired gene, as a particular cell will make some very specific types of mRNA.
• Formation of recombinant DNA

  • The DNA isolated by restriction endonucleases must be inserted into a vector that can carry it into a host cell.
• Vectors

  • Most commonly used vectors in genetic engineering are bacteria plasmids= small circular molecules of DNA separate from the chromosomal DNA that can replicate independently.
  • Once a plasmid gets into a new host cell it can combine with the host DNA to form recombinant DNA.
  • Plasmids are often chosen because they contain a marker gene. This gene enables scientists to determine that the bacteria have taken up the plasmid.
• • To insert a DNA fragment into a plasmid it must be cut open by the same restriction endonucleases. This results in the plasmid having complementary sticky ends to the sticky ends of the DNA fragment.
  • Once the complementary bases of the two sticky ends are lined up, the enzyme DNA ligase form phosphodiester bonds between the sugar and the phosphate groups on the two strands of DNA, joining them together.
• Second marker gene

  • Plasmids are given a second marker gene, which is used to show that the plasmid contains the recombinant gene.
  • The marker is often placed in the plasmid, the plasmid is then cut by a restriction enzymes within the marker gene to insert the desired gene.
  • If the gene is inserted successfully, the marker gene will not function.
  • Genes producing fluorescence or enzymes that change colour in a particular medium are used as maker genes.
• Transformation= the plasmid with the recombinant DNA must be transferred into the host cell.
• Transferring vectors

  1. Culture the bacterial cells and plasmids in a calcium rich solution and increase the temperature. This causes the bacterial membrane to become permeable and plasmids can enter.
  2. Another method is electroporation. A small electrical current is applied to the bacteria. This makes the bacteria very porous and so the plasmids move into the cells. Can also be used to get the DNA fragments directly into cells, the new DNA will pass through the cell membrane and the nuclear membrane to fuse with the nuclear DNA.
• Electrofusion in plants

  = Another way of producing genetically modified cells.

  • Tiny electrical currents are applied to the membranes of two different cells, this fuses the cell and nuclear membranes of the two different cells together to form a hybrid or polyploid cell, containing DNA from both.
• Electrofusion in animals

  • They don't fuse as easily because their membrane have different properties and polyploid animal cells don't usually survive in the body of a living organism.
  • Electrofusion is used in production of monoclonal antibodies. Are now used to identify pathogens in both animals and plants and in the treatment of a number of diseases.
• Engineering prokaryotes

  • Bacteria have been genetically modified to produce many useful substances to people.
  • Hormones like insulin and human growth hormone
  • Clotting factors for haemophiliacs, antibiotics, vaccines.
• Engineering plants

  • One method of genetically modifying plants use Agrobacterium tumefaciens, a bacterium that causes tumours in healthy plants.
  • A desired gene (for pesticide production, herbicide resistance or higher yield) is placed in the plasmid of A. Tumefaciens along with a marker gene. This is then carried directly into the plant cell DNA. The transgenic plant from a callus (made up of transformed cells which are undifferentiated) which is a mass of GM plant cells, each of which can be grown into a new transgenic plant.
• Engineering plants

  • Transgenic plants can also be produced by electrofusion.
  • The cells produced have chromosomes from both of the original cells and so are polyploid. The cells fused may be from similar or different species.
  • The main stages involve the removal of the plant cell wall by cellulases, electrofusion to form a new polyploid cell, the use of plant hormones to stimulate the growth of a new cell wall, followed by callus formation and the production of many cloned, transgenic plants.