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Retroviruses are a group of human viruses that include HIV. They are enclosed in a lipid membrane and their genetic material is double-stranded RNA. On infection this RNA is copied to DNA and the DNA is incorporated into the host’s chromosome. This means that the foreign genes are replicated into every daughter cell. After a certain time, the dormant DNA is switched on, and the genes are expressed in the host cells.
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Plant Tumours. This method has been used successfully to transform plant cells, which are perhaps the hardest to do. The gene is first inserted into the plasmid of a soil bacterium, and then plants are infected with the bacterium. The bacterium inserts the plasmid into the plant cells' chromosomal DNA and causes a "crown gall" tumour. These tumour cells can be cultured in the laboratory.
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Gene Gun. This technique fires microscopic gold particles coated with the foreign DNA at the cells using a compressed air gun. It is designed to overcome the problem of the strong cell wall in plant tissue.
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Micro-Injection. A cell is held on a pipette under a microscope and the foreign DNA is injected directly into the nucleus using an incredibly fine micro-pipette. Used where there are only a very few cells available, such as fertilised animal egg cells.
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. Vectors can be encased in liposomes, which are small membrane vesicles (see module 1). The liposomes fuse with the cell membrane (and sometimes the nuclear membrane too), delivering the DNA into the cell. This works for many types of cell, but is particularly useful for delivering genes to cell in vivo (such as in gene therapy).
GENETIC MARKERS
These are needed to identify cells that have successfully taken up a vector and so become transformed. With most of the techniques above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others. A common marker, used in plasmids, is a gene for resistance to an antibiotic such as tetracycline. Bacterial cells taking up this plasmid are resistant to this antibiotic. So if the cells are grown on a medium containing tetracycline all the normal untransformed cells (99%) will die. Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate.
REPLICA PLATING
Replica plating is a simple technique for making an exact copy of an agar plate. A pad of sterile cloth the same size as the plate is pressed on the surface of an agar plate with bacteria growing on it. Some cells from each colony will stick to the cloth. If the cloth is then pressed onto a new agar plate, some cells will be deposited and colonies will grow in exactly the same positions on the new plate. This technique has a number of uses, but the most common use in genetic engineering is to help solve another problem in identifying transformed cells. This problem is to distinguish those cells that have taken up a hybrid plasmid vector (with a foreign gene in it) from those cells that have taken up plasmids without the gene. This is where the second marker gene (for resistance to ampicillin) is used. If the foreign gene is inserted into the middle of this marker gene, the marker gene is disrupted and won't make its proper gene product. So cells with the hybrid plasmid will be killed by ampicillin, while cells with the normal plasmid will be immune to ampicillin. Since this method of identification involves killing the cells we want, we must first make a master agar plate and then make a replica plate of this to test for ampicillin resistance.
Once the colonies of cells containing the correct hybrid plasmid vector have been identified, the appropriate colonies on the master plate can be selected and grown on another plate.
POLYMERASE CHAIN REACTION (PCR)
Genes can be cloned by cloning the bacterial cells that contain them, but this requires quite a lot of DNA in the first place. PCR can clone (or amplify) DNA samples as small as a single molecule. It is a newer technique, having been developed in 1983 by Kary Mullis, for which discovery he won the Nobel prize in 1993. The polymerase chain reaction is simply DNA replication in a test tube. If a length of DNA is mixed with the four nucleotides (A, T, C and G) and the enzyme DNA polymerase in a test tube, then the DNA will be replicated many times.
- Start with a sample of the DNA to be amplified, and add the four nucleotides and the enzyme DNA polymerase.
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Normally (in vivo) the DNA double helix would be separated by the enzyme helicase, but in PCR (in vitro) the strands are separated by heating to 95°C for two minutes. This breaks the hydrogen bonds.
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DNA polymerisation always requires short lengths of DNA (about 20 bp long) called primers, to get it started. In vivo the primers are made during replication by DNA polymerase, but in vitro they must be synthesised separately and added at this stage. This means that a short length of the sequence of the DNA must already be known, but it does have the advantage that only the part between the primer sequences is replicated. The DNA must be cooled to 40°C to allow the primers to anneal to their complementary sequences on the separated DNA strands.
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The DNA polymerase enzyme can now extend the primers and complete the replication of the rest of the DNA. The enzyme used in PCR is derived from the thermophilic bacterium Thermus aquaticus, which grows naturally in hot springs at a temperature of 90°C, so it is not denatured by the high temperatures in step 2. Its optimum temperature is about 72°C, so the mixture is heated to this temperature for a few minutes to allow replication to take place as quickly as possible.
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Each original DNA molecule has now been replicated to form two molecules. The cycle is repeated from step 2 and each time the number of DNA molecules doubles. This is why it is called a chain reaction, since the number of molecules increases exponentially, like an explosive chain reaction. Typically PCR is run for 20-30 cycles.
PCR can be completely automated, so in a few hours a tiny sample of DNA can be amplified millions of times with little effort. The product can be used for further studies, such as cloning, electrophoresis, or gene probes. Because PCR can use such small samples it can be used in forensic medicine (with DNA taken from samples of blood, hair or semen), and can even be used to copy DNA from mummified human bodies, extinct woolly mammoths, or from an insect that's been encased in amber since the Jurassic period. One problem of PCR is having a pure enough sample of DNA to start with. Any contaminant DNA will also be amplified, and this can cause problems, for example in court cases.
Complementary DNA
Complementary DNA (cDNA) is DNA made from mRNA. This makes use of the enzyme reverse transcriptase, which does the reverse of transcription: it synthesises DNA from an RNA template. It is produced naturally by a group of viruses called the retroviruses (which include HIV), and it helps them to invade cells. In genetic engineering reverse transcriptase is used to make an artificial gene of cDNA as shown in this diagram.
Complementary DNA has helped to solve many problems in genetic engineering:
It makes genes much easier to find. There are some 70 000 genes in the human genome, and finding one gene out of this many is a very difficult (though not impossible) task. However a given cell only expresses a few genes, so only makes a few different kinds of mRNA molecule. For example the b cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene.
DNA Probes
These are used to identify and label DNA fragments that contain a specific sequence. A probe is simply a short length of DNA (20-100 nucleotides long) with a label attached. There are two common types of label used:
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a radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using photographic film (an autoradiograph).
- a fluorescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours.
Probes are always single-stranded, and can be made of DNA or RNA. If a probe is added to a mixture of different pieces of DNA (e.g. restriction fragments) it will anneal (base pair) with any lengths of DNA containing the complementary sequence. These fragments will now be labelled and will stand out from the rest of the DNA. DNA probes have many uses in genetic engineering:
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To identify restriction fragments containing a particular gene out of the thousands of restriction fragments formed from a genomic library. This use is described in shotguning below.
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To identify the short DNA sequences used in DNA fingerprinting.
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To identify genes from one species that are similar to those of another species. Most genes are remarkably similar in sequence from one species to another, so for example a gene probe for a mouse gene will probably anneal with the same gene from a human. This has aided the identification of human genes.
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To identify genetic defects. DNA probes have been prepared that match the sequences of many human genetic disease genes such as muscular dystrophy, and cystic fibrosis. Hundreds of these probes can be stuck to a glass slide in a grid pattern, forming a DNA microarray (or DNA chip). A sample of human DNA is added to the array and any sequences that match any of the various probes will stick to the array and be labelled. This allows rapid testing for a large number of genetic defects at a time.
SHOTGUNING
This is used to find one particular gene in a whole genome, a bit like finding the proverbial needle in a haystack. It is called the shotgun technique because it starts by indiscriminately breaking up the genome (like firing a shotgun at a soft target) and then sorting through the debris for the particular gene we want. For this to work a gene probe for the gene is needed, which means at least a short part of the gene’s sequence must be known.
Antisense Genes
These are used to turn off the expression of a gene in a cell. The principle is very simple: a copy of the gene to be switch off is inserted into the host genome the "wrong" way round, so that the complementary (or antisense) strand is transcribed. The antisense mRNA produced will anneal to the normal sense mRNA forming double-stranded RNA. Ribosomes can’t bind to this, so the mRNA is not translated, and the gene is effectively "switched off".
Electrophoresis
This is a form of chromatography used to separate different pieces of DNA on the basis of their length. It might typically be used to separate restriction fragments. The DNA samples are placed into wells at one end of a thin slab of gel (usually made of agarose) and covered in a buffer solution. An electric current is passed through the gel. Each nucleotide in a molecule of DNA contains a negatively-charged phosphate group, so DNA is attracted to the anode (the positive electrode). The molecules have to diffuse through the gel, and smaller lengths of DNA move faster than larger lengths, which are retarded by the gel. So the smaller the length of the DNA molecule, the further down the gel it will move in a given time. At the end of the run the current is turned off.
Unfortunately the DNA on the gel cannot be seen, so it must be visualised. There are three common methods for doing this:
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The gel can be stained with a chemical that specifically stains DNA, such as ethidium bromide. The DNA shows up as blue bands.
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The DNA samples at the beginning can be radiolabelled with a radioactive isotope such as 32P. Photographic film is placed on top of the finished gel in the dark, and the DNA shows up as dark bands on the film. This method is extremely sensitive.
- The DNA fragments at the beginning can be labelled with a fluorescent molecule. The DNA fragments show up as coloured lights when the finished gel is illuminated with invisible ultraviolet light.
DNA SEQUENCING
This means reading the base sequence of a length of DNA. Once this is known the amino acid sequence of the protein that the DNA codes for can also be determined, using the genetic code table. The sequence can also be compared with DNA sequences from other individuals and even other species to work out relationships.
DNA sequencing is based on a beautifully elegant technique developed by Fred Sanger, and now called the Sanger method.
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Label 4 test tubes labelled A, T, C and G. Into each test tube add: a sample of the DNA to be sequenced (containing many millions of individual molecules) a radioactive primer (so the DNA can be visualised later on the gel), the four DNA nucleotides and the enzyme DNA polymerase.
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In each test tube add a small amount of a special modified dideoxy nucleotide that cannot form a bond and so stops further synthesis of DNA. Tube A has dideoxy A (A*), tube T has dideoxy T (T*), tube C has dideoxy C (C*) and tube G has dideoxy G (G*). The dideoxy nucleotides are present at about 1% of the concentration of the normal nucleotides.
- Let the DNA polymerase synthesise many copies of the DNA sample. From time to time at random a dideoxy nucleotide will be added to the growing chain and synthesis of that chain will then stop. A range of DNA molecules will be synthesised ranging from full length to very short. The important point is that in tube A, all the fragments will stop at an A nucleotide. In tube T, all the fragments will stop at a T nucleotide , and so on.
- The contents of the four tubes are now run side by side on an electrophoresis gel, and the DNA bands are visualised by autoradiography. Since the fragments are now sorted by length the sequence can simply be read off the gel starting with the smallest fragment (just one nucleotide) at the bottom and reading upwards.
There is now a modified version of the Sanger method called cycle sequencing, which can be completely automated. The primers are not radiolabelled, but instead the four dideoxy nucleotides are fluorescently labelled, each with a different colour (A* is green, T* is red, C* is blue and G* is yellow). The polymerisation reaction is done in a single tube, using PCR-like cycles to speed up the process. The resulting mixture is separated using capillary electrophoresis, which gives good separation in a single narrow gel. The gel is read by a laser beam and the sequence of colours is converted to a DNA sequence by computer program (like the screenshot below). This technique can sequence an amazing 12 000 bases per minute.
Thousands of genes have been sequenced using these methods and the entire genomes of several organisms have also been sequenced. A huge project is underway to sequence the human genome, and it delivered a draft sequence in June 2000. The complete 3 billion base sequence should be complete by 2003. This information will give us unprecedented knowledge about ourselves, and is likely to lead to dramatic medical and scientific advances.
Applications of Genetic Engineering
We have now looked at some of the many techniques used by genetic engineers. What can be done with these techniques? By far the most numerous applications are still as research tools, and the techniques above are helping geneticists to understand complex genetic systems. Despite all the hype, genetic engineering still has very few successful commercial applications, although these are increasing each year. The applications so far can usefully be considered in three groups.
- Gene Products using genetically modified organisms (usually microbes) to produce chemicals, usually for medical or industrial applications
- New Phenotypes using gene technology to alter the characteristics of organisms (usually farm animals or crops)
- Gene Therapy using gene technology on humans to treat a disease
Gene Products
The biggest and most successful kind of genetic engineering is the production of gene products. These products are of medical, agricultural or commercial value. This table shows a few of the examples of genetically engineered products that are already available.
The products are mostly proteins, which are produced directly when a gene is expressed, but they can also be non-protein products produced by genetically-engineered enzymes. The basic idea is to transfer a gene (often human) to another host organism (usually a microbe) so that it will make the gene product quickly, cheaply and ethically. It is also possible to make "designer proteins" by altering gene sequences, but while this is a useful research tool, there are no commercial applications yet.
Since the end-product is just a chemical, in principle any kind of organism could be used to produce it. By far the most common group of host organisms used to make gene products are the bacteria, since they can be grown quickly and the product can be purified from their cells. Unfortunately bacteria cannot not always make human proteins, and recently animals and even plants have also been used to make gene products. In neither case is it appropriate to extract the product from their cells, so in animals the product must be secreted in milk or urine, while in plants the product must be secreted from the roots. This table shows some of the advantages and disadvantages of using different organisms for the production of genetically-engineered gene products.
We’ll look at some examples in detail.
Human Insulin
Insulin is a small protein hormone produced by the pancreas to regulate the blood sugar concentration. In the disease insulin-dependent diabetes the pancreas cells don’t produce enough insulin, causing wasting symptoms and eventually death. The disease can be successfully treated by injection of insulin extracted from the pancreases of slaughtered cows and pigs. However the insulin from these species has a slightly different amino acid sequence from human insulin and this can lead to immune rejection and side effects.
The human insulin gene was isolated, cloned and sequenced in the 1970s, and so it became possible to insert this gene into bacteria, who could then produce human insulin in large amounts. Unfortunately it wasn’t that simple. In humans, pancreatic cells first make pro-insulin, which then undergoes post-translational modification to make the final, functional insulin. Bacterial cells cannot do post-translational modification. Eventually a synthetic cDNA gene was made and inserted into the bacterium E. coli, which made pro-insulin, and the post-translational conversion to insulin was carried out chemically. This technique was developed by Eli Lilly and Company in 1982 and the product, "humulin" became the first genetically-engineered product approved for medical use.
In the 1990s the procedure was improved by using the yeast Saccharomyces cerevisiae instead of E. coli. Yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin. However another company has developed a method of converting pig insulin into human insulin by chemically changing a few amino acids, and this turns out to be cheaper than the genetic engineering methods. This all goes to show that genetic engineers still have a lot to learn.
Bovine Somatotrophin (BST)
This is a growth hormone produced by cattle. The gene has been cloned in bacteria by the company Monsanto, who can produce large quantities of BST. in the USA cattle are often injected with BST every 2 weeks, resulting in a 10% increase in mass in beef cattle and a 25% increase in milk production in dairy cows. BST was tested in the UK in 1985, but it was not approved and its use is currently banned in the EU. This is partly due to public concerns and partly because there is already overproduction of milk and beef in the EU, so greater production is not necessary.
Rennin
Rennin is an enzyme used in the production of cheese. It is produced in the stomach of juvenile mammals (including humans) and it helps the digestion of the milk protein caesin by solidifying it so that is remains longer in the stomach. The cheese industry used to obtain its rennin from the stomach of young calves when they were slaughtered for veal, but there are moral and practical objections to this source. Now an artificial cDNA gene for rennin has been made from mRNA extracted from calf stomach cells, and this gene has been inserted into a variety of microbes. The rennin extracted from these microbes has been very successful and 90% of all hard cheeses in the UK are made using microbial rennin. Sometimes (though not always) these products are labelled as "vegetarian cheese".
AAT (α-1-antitrypsin)
AAT is a human protein made in the liver and found in the blood. As the name suggests it is an inhibitor of protease enzymes like trypsin and elastase. There is a rare mutation of the AAT gene (a single base substitution) that causes AAT to be inactive, and so the protease enzymes to be uninhibited. The most noticeable effect of this in the lungs, where elastase digests the elastic tissue of the alveoli, leading to the lung disease emphysema. This condition can be treated by inhaling an aerosol spray containing AAT so that it reaches the alveoli and inhibits the elastase there.
AAT for this treatment can be extracted from blood donations, but only in very small amounts. The gene for AAT has been found and cloned, but AAT cannot be produced in bacteria because AAT is a glycoprotein, which means it needs to have sugars added by post translational modification. This kind of modification can only be carried out by animals (because they have a golgi body), and AAT is now produced by genetically-modified sheep. In order to make the AAT easy to extract, the gene was coupled to a promoter for the milk protein b-lactoglubulin. Since this promoter is only activated in mammary gland cells, the AAT gene will only be expressed in mammary gland cells, and so will be secreted into the sheep's milk. This makes it very easy to harvest and purify without harming the sheep. The first transgenic sheep to produce AAT was called Tracy, and she was produced in Edinburgh in 1993. This is how Tracy was made:
New Phenotypes
This means altering the characteristics of organisms by genetic engineering. The organisms are usually commercially-important crops or farm animals. It can be seen as a high-tech version of selective breeding, which has been used by humans to alter and improve their crops and animals for at least 10 000 years. Nevertheless GMOs have turned out to be a highly controversial development. We don’t study any of these in detail, but this table gives an idea of what is being done.
Gene Therapy
This is perhaps the most significant, and most controversial kind of genetic engineering. It is also the least well-developed. The idea of gene therapy is to genetically alter humans in order to treat a disease. This could represent the first opportunity to cure incurable diseases. Note that this is quite different from using genetically-engineered microbes to produce a drug, vaccine or hormone to treat a disease by conventional means. Gene therapy means altering the genotype of a tissue or even a whole human.
Cystic Fibrosis (you must learn this one!)
Cystic fibrosis (CF) is the most common genetic disease in the UK, affecting about 1 in 2500. It is caused by a mutation in the gene for protein called CFTR (Cystic Fibrosis Transmembrane Regulator). The gene is located on chromosome 7, and there are actually over 300 different mutations known, although the most common mutation is a deletion of three bases, removing one amino acid out of 1480 amino acids in the protein. CFTR is a chloride ion channel protein found in the cell membrane of epithelial (lining) tissue cells, and the mutation stops the protein working, so chloride ions cannot cross the cell membrane.
Chloride ions build up inside these cells, which cause sodium ions to enter to balance the charge, and the increased concentration of the both these ions inside the epithelial cells decreases the osmotic potential. Water is therefore retained inside the cells, which means that the mucus secreted by these cells is drier and more sticky than normal. This sticky mucus block the tubes into which it is secreted, such as the small intestine, pancreatic duct, bile duct, sperm duct, bronchioles and alveoli.
These blockages lead to the symptoms of CF: breathlessness, lung infections such as bronchitis and pneumonia, poor digestion and absorption, and infertility. Of these symptoms the lung effects are the most serious causing 95% of deaths. CF is always fatal, though life expectancy has increased from 1 year to about 20 years due to modern treatments. These treatments include physiotherapy many times each day to dislodge mucus from the lungs, antibiotics to fight infections, DNAse drugs to loosen the mucus, enzymes to help food digestion and even a heart-lung transplant.
Given these complicated (and ultimately unsuccessful) treatments, CF is a good candidate for gene therapy, and was one of the first diseases to be tackled this way. The gene for CFTR was identified in 1989 and a cDNA clone was made soon after. The idea is to deliver copies of this good gene to the epithelial cells of the lung, where they can be incorporated into the nuclear DNA and make functional CFTR chloride channels. If about 10% of the cells could be corrected, this would cure the disease.
Two methods of delivery are being tried: and , both delivered with an aerosol inhaler, like those used by asthmatics. Clinical trials are currently underway, but as yet no therapy has been shown to be successful.
The Future of Gene Therapy
Gene therapy is in its infancy, and is still very much an area of research rather than application. No one has yet been cured by gene therapy, but the potential remains enticing. Gene therapy need not even be limited to treating genetic diseases, but could also help in treating infections and environmental diseases:
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White blood cells have be genetically modified to produce tumour necrosis factor (TNF), a protein that kills cancer cells, making these cells more effecting against tumours.
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Genes could be targeted directly at cancer cells, causing them to die, or to revert to normal cell division.
- White blood cells could be given antisense genes for HIV proteins, so that if the virus infected these cells it couldn’t reproduce.
It is important to appreciate the different between somatic cell therapy and germ-line therapy.
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Somatic cell therapy means genetically altering specific body (or somatic) cells, such as bone marrow cells, pancreas cells, or whatever, in order to treat the disease. This therapy may treat or cure the disease, but any genetic changes will not be passed on their offspring.
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Germ-line therapy means genetically altering those cells (sperm cells, sperm precursor cell, ova, ova precursor cells, zygotes or early embryos) that will pass their genes down the "germ-line" to future generations. Alterations to any of these cells will affect every cell in the resulting human, and in all his or her descendants.
Germ-line therapy would be highly effective, but is also potentially dangerous (since the long-term effects of genetic alterations are not known), unethical (since it could easily lead to eugenics) and immoral (since it could involve altering and destroying human embryos). It is currently illegal in the UK and most other countries, and current research is focussing on somatic cell therapy only. All gene therapy trials in the UK must be approved by the Gene Therapy Advisory Committee (GTAC), a government body that reviews the medical and ethical grounds for a trial. Germ-line modification is allowed with animals, and indeed is the basis for producing GMOs.
Biology notes mod 2 Genetic Engineering