Genetic engineering

Isolated the desired gene

• Most common technique is the use of restriction endonucleases to cut the required gene from the DNA
• Some make a clean, blunt-ended cut in the DNA but others cut the DNA strands unevenly leaving sticky ends
• Sticky ends make it easier to insert the desired gene into the DNA of a different organism
• Another technique involves isolating the mRNA for the desired gene and using reverse transcriptase to produce a single strand of complementary DNA
• ^^ easier to identify the desired gene

The formation of recombinant DNA

Vectors

• Most common vectors are bacterial plasmids (small circular molecules of DNA that can replicate independently)
• Once the plasmid gets into a host cell it can combine with the host DNA to form recombinant DNA
• The plasmids used as vectors are often chosen because they contain a marker gene which allow the scientists to see when the plasmid has been taken up
• The same restriction endonuclease is used to cut open the DNA of the plasmid so that the sticky ends of the isolated gene will match up with the hole
• Once the isolated gene and plasmid DNA have matched up by complementary base pairing, DNA ligase forms the phosphodiester bonds to join the DNA strands together
• A second marker is often added alongside the desired gene
• If the DNA is inserted successfully, the additional marker gene won't function (if its a fluorescent one then the bacterium will not fluoresce if its been engineered successfully)

Transferring the vector

• Transformation = transfer of the plasmid with the recombinant DNA into the host cell
• One way is to culture the bacterial cells in a calcium-rich solution at a high temperature --> increases the permeability of bacterial membranes so the plasmids enter
• Another is electroporation where a small electric current is applied to the bacteria to make the membranes porous and allow the plasmids in
• ^^ can be used to get DNA fragments directly into eukaryotic cells (new DNA will pass through cell and nuclear membrane to fuse with the nuclear DNA)
• ^^^ effective but cannot be 100% controlled and damages the membrane permantently 

      Electrofusion

• Tiny electric currents applied to membranes of two different cells
• Fuses cell and nuclear membranes of the two cells to form a hybrid or polyploid cell with DNA from both
• Used to produce GM plants
• Not used for animal cells --> different membrane structures
• Important in the production of monoclonal antibodies --> combination of a plasma cell and a tumour cell
• Used to identify pathogens in both plants and animals and in treatment of cancers

Engineering in different organisms

Prokaryotes

• Genetically modified to produce substances that are useful to humans like hormones. clotting factors, antibiotics, pure vaccines and enzymes for industry

Plants

• Use of bacterium that causes tumours in healthy plants
• Desired gene (e.g. pesticide production) placed in the plasmid of the bacterium with a marker gene (for antibiotic resistance or fluorescence) 
• This is the carried directly into the plant cell DNA
• Transgenic cells form a callus (mass of GM plant cells) each of which can be grown into a new GM plant
• OR by electrofusion --> cells produced are polyploid (have chromosomes from both original cells)
  1. Removal of plant cell walls by cellulases
  2. Electrofusion to form a new polyploid cell
  3. Use of plant hormones to stimulate growth of new cell wall
  4. Callus formation
  5. Production of many transgenic plants

Animals

• Harder to modify animal cells
• Membranes less easy to manipulate
• Important technique to allow animal cells to make medically important proteins and to try and cure genetic diseases