Genetic Engineering

2. The process of genetic engineering

The general procedure for inserting a new gene into a host organism has the same five steps every time.

Step 1: Isolate the desired gene

The gene that codes for the desired protein is first found within the source organism's DNA. A specific restriction enzyme then cuts the gene out. Restriction enzymes are remarkable: each one only cuts the DNA at a specific base sequence (called a recognition site), so you can choose the right enzyme to cut out exactly the gene you want.

When a restriction enzyme cuts double-stranded DNA, it leaves short overhangs of unpaired bases on each cut end. These overhangs are called sticky ends because they will pair up with any other piece of DNA that has matching complementary bases.

Step 2: Prepare the vector

The vector is usually a bacterial plasmid (a small circular piece of DNA found inside bacteria, separate from the main chromosome). The same restriction enzyme is used to cut the plasmid open. Because it is the same enzyme, the plasmid ends up with complementary sticky ends to the desired gene.

Using the same enzyme to cut both the gene and the plasmid is crucial. It is what guarantees the sticky ends will match and pair up.

Step 3: Join the gene to the vector

The cut gene and the opened plasmid are mixed together. Their sticky ends find each other and pair up using the base-pairing rules (A with T, G with C). The enzyme DNA ligase then seals the joints, forming a single complete molecule of recombinant DNA (DNA made from two different sources joined together).

Step 4: Insert the vector into the host cell

The recombinant plasmid is added to a culture of host cells (usually bacteria). Some of the cells take the plasmid in (a process called transformation). The host cell now contains the new gene and can use it to make the new protein.

Step 5: Grow the host cells in a fermenter

The transformed bacteria are placed in a large industrial fermenter along with their nutrients and the right temperature, pH and oxygen conditions. They multiply rapidly. Every time a bacterium divides, the plasmid is copied too, so every new bacterium contains the gene and makes the protein. Within a few days the fermenter contains millions of bacteria all producing the desired protein, which can then be extracted and purified.

3. GM bacteria producing human insulin

The classic example of genetic engineering, and the first to be commercialised in 1982, is the production of human insulin for treating people with type 1 diabetes. Before genetic engineering, diabetics had to use insulin extracted from cow or pig pancreases, which was expensive, in short supply, and slightly different from human insulin (some patients developed allergic reactions).

Step by step

Applying the general five-step process specifically to insulin:

1. Locate the human insulin gene on human chromosome 11.
2. Use a specific restriction enzyme to cut the gene out of human DNA. The gene now has sticky ends.
3. Use the same restriction enzyme to cut open a bacterial plasmid. The plasmid now has matching sticky ends.
4. Mix the gene with the cut plasmid. The sticky ends pair up by complementary base pairing.
5. DNA ligase seals the joints, forming a recombinant plasmid containing the human insulin gene.
6. The recombinant plasmid is inserted into bacterial cells (usually Escherichia coli).
7. The bacteria are grown in industrial fermenters under controlled conditions (temperature, pH, oxygen, nutrients). They multiply rapidly.
8. Each bacterium reads the human insulin gene and produces human insulin protein from it. (The bacterium's own ribosomes do not care that the gene is human; they read the code the same way they would for any bacterial gene.)
9. The insulin is extracted and purified from the fermenter and packaged as medical-grade insulin.

Why bacteria are ideal for this

Bacteria are the workhorse of genetic engineering for several reasons:

• They have plasmids, which are easy to remove, modify and put back. This is the natural vector for engineering bacteria.
• They reproduce extremely fast (one cell can become billions in a day), so you can rapidly produce large quantities of the modified organism.
• They use the same genetic code as humans (and every other organism), so they can read and translate human genes correctly.
• They don't raise the same ethical concerns as engineering animals or plants. There is no animal welfare issue with bacteria.
• They are cheap and easy to grow.

Insulin today

Today, almost all of the world's medical insulin is made by GM bacteria using exactly this process. The insulin produced is chemically identical to human insulin, with no risk of allergic reaction. It can be made in unlimited quantities at low cost. This was one of the great medical successes of the late 20th century.

Other proteins made by GM bacteria include human growth hormone, blood-clotting factors for haemophilia patients, and several vaccines.

4. GM crops

Genetic engineering of plants has produced genetically modified crops with useful new traits.

Common GM traits in crops

Advantages of GM crops

Disadvantages and concerns

The scientific consensus is that current GM crops are safe to eat. The bigger debates are about social, economic and environmental impacts rather than direct health risks.

5. Transgenic animals

Animals can also be genetically engineered. The process is more complex than for bacteria because animals are multicellular and the gene must be inserted at the right early stage of development to end up in every cell of the adult.

Transgenic animals producing proteins for medicine

One major use of transgenic animals is producing useful proteins in their milk:

1. The gene for a useful human protein (e.g. a blood-clotting factor) is inserted into the DNA of a fertilised animal egg, typically a goat, sheep or cow.
2. The egg is implanted into a surrogate mother and develops normally into an animal that carries the gene.
3. The animal grows up and starts producing milk. The human protein is secreted into the milk along with normal milk proteins.
4. The milk is collected and the human protein is purified out.

This approach can make proteins that are too large or complex for bacteria to produce. It has been used to make:

• Alpha-1-antitrypsin (used to treat a rare lung disorder)
• Human blood-clotting factors for haemophilia patients
• Antithrombin (used during surgery to prevent dangerous clots)

The animals live normal lives; they are just very expensive milk producers.

Other uses of transgenic animals

• GM mice are widely used in medical research. Specific human disease genes can be put into mice so that researchers can study the disease and test possible treatments.
• Transgenic fish have been engineered to grow faster (e.g. AquAdvantage salmon, approved in the USA and Canada).
• GM mosquitoes carrying a "self-limiting" gene have been released in some areas to try to suppress mosquito populations that spread diseases like dengue and Zika.

6. Cloning of animals

Cloning means producing genetically identical copies of an organism. Cloning of plants by tissue culture / micropropagation was covered in topic 17. Cloning of animals is much harder because animal cells, unlike plant cells, lose their ability to develop into a whole organism very early in life. Only stem cells in the embryo can do this.

There are two main techniques for cloning animals.

Adult cell cloning (somatic cell nuclear transfer)

This is the technique that produced Dolly the sheep in 1996, the world's first mammal cloned from an adult cell. The five steps:

1. Take an unfertilised egg cell from a female of the species. Using a fine needle, remove its nucleus. The egg is now enucleated (it has no DNA of its own).
2. Take a body cell (any normal somatic cell, often a skin or udder cell) from the adult animal you want to clone. This cell contains the full diploid set of chromosomes.
3. Insert the diploid nucleus from the body cell into the enucleated egg. The egg now contains DNA identical to the adult animal.
4. Give the cell a mild electric shock to fool it into thinking it has been fertilised. The cell starts to divide by mitosis, forming an embryo.
5. Once the embryo has reached a small ball of cells, implant it into the uterus of a surrogate mother. The embryo develops normally and is eventually born.

The newborn is a genetic clone of the animal that supplied the body cell, with the same DNA in every cell. The surrogate mother contributes nothing genetically; she just carries the pregnancy.

Dolly the sheep

In 1996, scientists at the Roslin Institute in Scotland used this technique on sheep:

• DNA donor: a 6-year-old Finn Dorset ewe (white-faced)
• Egg donor: a Scottish Blackface ewe
• Surrogate mother: another Scottish Blackface ewe

Dolly was born with a white face like the DNA donor, not black like the surrogate, proving she was a true clone of the DNA donor and not a normal offspring of the surrogate. She lived to age 6 and had several lambs of her own. Dolly's success started the whole modern field of mammalian cloning.

Embryo cloning (embryo splitting)

A simpler technique called embryo cloning has been used in livestock breeding for decades:

1. Two parent animals with the desired traits are mated normally, producing a fertilised egg (zygote).
2. The zygote divides into an early embryo (a ball of identical stem cells).
3. The embryo is divided into smaller groups of cells, each of which is genetically identical.
4. Each piece is implanted into a separate surrogate mother.
5. Each pregnancy results in a clone of the original embryo, so all the offspring are genetically identical to each other (but not identical to either parent, since they come from a normal fertilised egg).

This is how naturally identical twins form: a single fertilised egg accidentally splits into two embryos that develop separately. Embryo cloning just does it on purpose, with more pieces, in more surrogates.

Uses of animal cloning

• Multiplying valuable livestock: one prize bull or top dairy cow can produce thousands of genetically identical offspring through cloning
• Producing transgenic animals at scale: once one transgenic animal has been engineered, cloning makes many more identical copies, all producing the same useful protein
• Conservation of endangered species: cloning could help restore populations of nearly-extinct animals
• Preserving extinct species: there is research into using preserved DNA to clone extinct species (woolly mammoths, dodos), though no successful cases yet
• Medical research: cloned animals provide genetically uniform test subjects, removing variation between individuals as a confounding factor

Limitations and risks

• Very low success rate: only a few percent of cloning attempts produce a healthy live birth. It took 277 attempts to produce Dolly.
• Health problems in clones: cloned animals often have shortened lifespans, premature ageing, weakened immune systems, and various other problems
• Loss of genetic variation: a herd of clones is genetically uniform, so vulnerable to any disease the clones cannot resist
• Ethical concerns (covered below)

7. Comparison with selective breeding

Genetic engineering and selective breeding both aim to produce organisms with useful new traits. They achieve this in very different ways.

The two techniques can be combined. Genetic engineering can introduce a useful gene; selective breeding can then improve other characteristics over generations.

8. Ethical and safety concerns

Genetic engineering raises serious ethical and safety questions that scientists, governments and the public have been debating for decades. There are no simple answers.

Safety concerns

• Could the engineered organism harm human health? GM food has been eaten by millions of people for over two decades with no clear evidence of harm, but long-term effects are uncertain.
• Could engineered genes spread to wild populations? Pollen from GM crops can fertilise wild relatives. Genes for herbicide resistance, for example, could spread to weeds, making them harder to kill.
• Could engineered organisms unbalance ecosystems? Bt crops kill the target pests but might also harm non-target species like butterflies that share habitat with crops.
• Could pests evolve resistance? Yes. Insects exposed to Bt crops will be under intense selection for any Bt-resistance allele. Resistance has already been observed in some places.
• Risk of escape: GM fish or insects released could establish wild populations that crowd out natives.

Ethical concerns

• Playing God: some people feel humans should not deliberately alter the genome of any organism, especially across species boundaries. This concern often has religious roots.
• Animal welfare: transgenic animals may suffer from health problems caused by their engineered genes; cloned animals often have shorter lifespans and weaker health.
• Patents and ownership of life: companies can patent the GM organisms they create, treating living things as private property. Critics argue life should not be owned by corporations.
• Inequality: GM seeds and patented livestock benefit large agribusinesses more than small farmers. Some farmers become dependent on annual seed purchases from a single supplier.
• Reduced choice: cross-contamination between GM and non-GM farms can mean farmers and consumers lose the choice to avoid GM products.
• Human gene editing: the same technology can in principle be used on human embryos. In 2018 a Chinese scientist produced the first gene-edited human babies, triggering global condemnation and tighter regulation. The line between curing disease and "designer babies" is murky.

Reasons for cautious optimism

Genetic engineering has also brought real benefits:

• Insulin for millions of diabetics
• Vaccines produced quickly and at low cost
• Vitamin-enriched crops that could prevent millions of cases of malnutrition
• Drought-tolerant crops for areas facing climate change
• Medical research tools that have led to important discoveries
• Conservation potential for endangered species

The regulation question

Different countries take very different approaches:

• The USA treats GM crops mostly like any other crop, with limited labelling.
• The European Union is more cautious: GM crops must be labelled, and many are banned from growing in EU countries.
• The UK is currently relaxing some rules to allow more gene-edited crops.
• Many countries in Africa and Asia have a patchwork of approaches.

Whether genetic engineering is mostly a force for good or for harm depends partly on how carefully it is regulated, who benefits from the new technology, and how transparently companies and governments handle the risks.

9. The full toolkit of biological resource techniques

This is the last topic in the biology syllabus. Bringing together everything from topics 12, 17 and 18, modern biotechnology offers a toolkit of techniques for producing useful plants, animals and proteins:

Each technique has its place. Together, they have completely transformed how humans produce food, medicines and industrial materials, and they continue to develop rapidly. The biggest challenges going forward are not really technical but ethical, regulatory and social: how to deploy these powerful tools wisely, equitably, and with appropriate caution.