Gene Transformations


The genetic code is universal, meaning that for every living organism the same codons code for the same amino acids (there are a few rare exceptions)

This means that the genetic information from one organism could be translated by another (i.e. it is theoretically transferable) 

The process by which genes are transferred to create genetically modified organisms with new traits involves a number of key steps:


DNA Extraction

  • The DNA from an organism of interest can be isolated and extracted via centrifugation
  • This involves spinning a mixture of cells to separate out the heavier components (such as the nuclei)
  • The DNA within the nuclei can then be precipitated out of solution and then purified
  • Finally, the gene of interest can be specifically amplified via the polymerase chain reaction (PCR)
  • DNA sequences can also be generated from mRNA transcripts using the viral enzyme reverse transcriptase


Digestion

  • In order to incorporate the gene of interest into a vector for delivery and expression, the sequence must first be digested with restriction enzymes
  • Cutting with certain restriction enzymes at specific recognition sites may generate short sequence overhangs ("sticky ends") that allow the DNA construct to fit into a vector


Digesting with a Restriction Endonuclease to Generate 'Sticky Ends'

Ligation

  • The gene of interest is inserted into a vector (a gene delivery mechanism) that was cut with the same restriction enzymes
  • This allows the sticky ends of the gene and the vector to overlap via complementary base pairing
  • The gene of interest and the vector are spliced together by the enzyme DNA ligase to create a recombinant construct
  • There are two main types of vectors used as gene delivery systems in most modern recombinant techniques:
    • Plasmids:  Circular molecules of DNA (isolated from bacteria) that can exist and replicate independently of the host genome 
    • Viruses:  Viruses may integrate their genetic material into the host genome, potentially allowing for more stable expression 


Expression

  • The recombinant construct containing the gene of interest is introduced into an appropriate host cell / organism
  • This process is called transfection in eukaryotes and transformation in prokaryotes
  • There are a number of ways via which this gene delivery can be mediated, including:
    • Heat-shocking  (briefly destabilises cell membrane to allow vector entry)
    • Electroporation  (brief electrical shock make temporary holes in the cell membrane)
    • Particle bombardment  (DNA-coated particles are shot into cells)
    • Transduction  (viral delivery of DNA into cell)
  • The transgenic cells will hopefully produce the desired trait encoded by the gene of interest (expression)
  • Antibiotic resistant genes are sometimes included in the engineered vector as a marker - only those cells that have successfully taken up the recombinant construct will be able to grow on a medium containing a specific antibiotic
  • This antibiotic selection will leave behind in culture only the cells that contain the recombinant construct - the product can then be isolated from these host cells and purified in order to generate sufficient yield


Treating Haemophilia via the Isolation of Human Factor IX Clotting Protein from Transgenic Sheep Milk


Examples of Genetic Modification

 Crops

1.  Engineering crops to extend shelf life of fresh produce

  • Tomatoes (Flavr Savr) have been engineered to have an extended keeping quality by switching off the gene for ripening and thus delaying the natural process of softening of fruit


2.  Engineering of crops to provide protection from insects

  • Maize crops (Bt corn) have been engineered to be toxic to the corn borer by introducing a toxin gene from a bacterium (Bacillus thuringiensis


Animals

1.  Engineering animals to enhance production

  • Sheep produce more wool when engineered with the gene for the enzyme responsible for the production of cysteine - the main amino acid in the keratin protein of wool


2.  Engineering animals to produce desired products

  • Sheep engineered to produce human alpha-1-antitrypsin in their milk can be used to help treat individuals suffering from hereditary emphysema