Factor VIII, also known as antihemophilic factor or AHF, is indicated for the treatment of patients suffering from hemophilia A, an inherited disorder in which the blood clotting protein Factor VIII is deficient or abnormal. Affected persons are unable to form blood clots normally and therefore risk serious and life-threatening bleeding episodes. Replacement therapy with Factor VIII corrects the defect temporarily but must be given by intravenous infusion, in many cases daily or more often.
Transformation
The first step in transformation is to select a piece of DNA to be inserted into a vector. In the case of insulin, the gene needed is from a human chromosome from a pancreatic cell. The second step is to isolate that gene; there are two methods of doing this:
- The first method involves cutting the gene from the rest of the DNA using restriction endonuclease enzymes; the required gene is located using electrophoresis. This works on the principle that if the same DNA is treated with the same restriction enzyme, the same fragments will be produced every time; these fragments will all be different lengths because the enzyme only cuts at certain sequences of bases. The DNA fragments are placed at the cathode end of a gel plate and an electric current is passed through the gel. The DNA fragments move through the gel towards the anode; the smallest fragments pass through the gel the easiest and so the smaller fragments travel faster and move further in a given time. The different distances that the fragments of DNA move produce invisible banding; this banding can be revealed by staining the DNA.
- The second method of isolation uses reverse transcription to make a copy of the gene. This is the opposite of normal transcription, as in this process a sequence of DNA is copied from a messenger RNA template. mRNA is obtained from cells from the human pancreas and used as a template to produce a complementary single strand of DNA from free nucleotides. This process is catalysed by the enzyme reverse transcriptase. The single strand of DNA is now made double stranded from free nucleotides and the enzyme DNA polymerase.
The advantages of using this method as opposed to the first isolation method is that each active cell will have many copies of the mRNA so it should be easier to find the single copy of the DNA gene in each cell. Also, the length of the mRNA strand corresponds exactly to the DNA gene, and does not need to be cut out of a longer piece. Another reason why the second method is more effective is that it is difficult to use genes form eukaryotic cell for use in prokaryotic cells when using restriction enzymes to cut the gene form the DNA chain. This is due to the presence of introns which, in eukaryotic cells, are edited out when proteins are made; prokaryotic cells however, do not edit them out and the wrong polypeptides may be synthesized. The second method uses mRNA in which the introns have already removed.
The second stage of transformation concerns inserting the gene into a vector; this is usually a molecule of DNA which carries the gene into a host cell. The most common forms of vectors are plasmids and phages (see phage introduction method).
Plasmids are highly suitable as vectors because they can move in and out of bacteria very easily and the plasmid DNA replicates when the bacteria reproduce. The isolated gene is joined to the plasmid DNA by means of ligation, this is catalysed by the enzyme DNA ligase. The vector is now inserted into the host cell. Firstly, the plasmids from the bacterial cells which are to act as the hosts are removed and the recombinant plasmids are incubated with the host bacterial cells. Various factors affect the rate at which plasmids enter the host bacteria such as: calcium ion concentration and temperature and electrical shock. Host cells that a have been entered by the recombinant plasmid are said to be transformed. The insert contains a selectable marker which allows for identification of recombinant molecules. An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant.
All transformed host cells must now be cloned, commercially; this is done in large industrial fermenters. Once a substantial amount of the bacteria has been produced, the insulin that they make through protein synthesis, which is chemically identical to human insulin, is extracted and purified for commercial use.
Non-Bacterial Transformation
This is a process very similar to Transformation, which was described above. The
only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.
In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.
Phage Introduction
Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packaging of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.
The various methods of using recombinant in medicine vary according the substance which is intended to be made and the type of bacteria that is to act as a host cell. Recombinant DNA or genetic engineering is now commonly used in agriculture and cattle breeding to combine positive features of various species. One of the main goals of genetic engineers is to produce a plant with nitrogen-fixing bacteria in its roots. This way it would be able to supply its own nitrates and have a vastly improved growth rate. Despite this however genetic engineering is not universally approved as there are a number of moral issues that have been raised as a result of its manipulating nature.
