Brent Cornell

    
A gene determines a particular trait by encoding for a specific polypeptide in a given organism

Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome

The transfer of genes between species is called gene modification, and the new organism created is called a transgenic

Genetic Modification: Bacteria Producing Human Insulin

    
The process of gene transfer can be summarised in four key steps:

1. Isolation of gene and vector (by PCR)
2. Digestion of gene and vector (by restriction endonuclease)
3. Ligation of gene and vector (by DNA ligase)
4. Selection and expression of transgenic construct

Step 1:  Isolating gene and vector

• DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
• The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
• Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
  
  
• A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
• Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression
• These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters) 
• Other types of vectors include modified viruses and artificial chromosomes

Common Features of a Typical Plasmid Vector

Step 2:  Digestion with Restriction Enzymes

• In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites
• Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
• Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from re-annealing without the desired insert

‘Sticky End’ vs ‘Blunt End’ Restriction Enzymes

Step 3:  Ligation of Vector and Insert

• The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
• This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
• The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
• DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond

Formation of a Recombinant Construct

Step 4:  Selection and Expression

• The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism
• This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)
• Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct
• The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic
• Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest

Isolation of Transgenic Cells via Antibiotic Selection