Genetic Engineering: Vectors
- Vectors are used to transfer the desired genes into a foreign cell
- Plasmids are the most commonly used vector but viruses and liposomes (a small vesicle with a phospholipid layer) can also be used to transfer genes
Plasmids
- Plasmids are small, circular rings of double-stranded DNA
- They occur naturally in bacteria, but can also been found in archaea and eukaryotic organisms (eg. yeast and fungi) and can contain genes for antibiotic resistance
- Plasmids are used as they can self replicate
- A plasmid is used to transfer the desired gene to a new organism
- To insert the desired gene into the circular DNA of the plasmid it is ‘cut’ open. The same restriction endonuclease that was used to isolate the desired gene is used to ‘cut’ open the plasmid. This results in the plasmid having complementary sticky ends to the sticky ends on the desired gene fragment
- DNA ligase forms phosphodiester bonds between the sugar-phosphate backbone of the DNA fragment and the plasmid to form a recombinant plasmid (a closed circle of double-stranded DNA containing the desired gene)
- Scientists can modify bacterial plasmids or artificially produce them. One benefit of this is that the plasmids can have one or more marker genes so that cells that have the recombinant plasmids can be identified
- Plasmids are transferred into host cells (usually bacteria) by a process called transformation. Only a small proportion of bacteria will become transformed and therefore markers are used to identify these. Transformation can occur by:
- Bathing the plasmids and bacteria in an ice-cold calcium chloride solution and then briefly incubating at 40°C. This makes the bacteria membrane permeable
- Electroporation - where the bacteria is given a small electrical shock making the membranes very porous (this technique can be used to get DNA fragments into eukaryotic cells)
A plasmid is genetically engineered with the desired gene
Viruses
- Viruses are commonly used as vectors in the process of gene therapy, which is currently used to treat genetic diseases such as cystic fibrosis
- The viruses are genetically modified to carry non-mutated genes into host cells
- Different types of viruses have been used; retroviruses, lentiviruses and adeno-associated viruses
An adenovirus is genetically engineered with the desired gene. Retroviruses have also been used as viral vectors however there have been side-effects with their use
Liposomes
- Liposomes are small spherical vesicles with a phospholipid layer
- These vesicles can also be used in gene therapy to carry non-mutated genes into host cells
- The advantage of using liposomes as a vector is that they can fuse with the cell surface membrane
A liposome is genetically engineered to carry a plasmid that contains the desired gene
Exam Tip
Remember two enzymes are used in the preparation of a plasmid vector – restriction endonucleases and DNA ligase. Also the same restriction endonuclease must be used to ‘cut’ open the plasmid as was used to isolate the desired gene.
