Insulin and vaccine production
Insulin controls blood sugar. Earlier it was taken from the pancreas of slaughtered cattle and pigs. Some patients were allergic to this animal insulin, and supply was limited.
Insulin is made in the body as pro-insulin: chains A and B plus an extra piece called the C-peptide. The C-peptide is removed in the mature hormone. In 1983 the American company Eli Lilly made human insulin by a clever method: they prepared two DNA pieces for chain A and chain B, put each into a plasmid of E. coli, got the chains made separately, purified them and joined them with disulfide bonds. This insulin is called humulin. No C-peptide step was needed.
Recombinant vaccines
A vaccine teaches the immune system to recognise a germ. Instead of using a whole germ, we can put only the gene for one surface protein (the antigen) into yeast or bacteria. The protein they make is used as the vaccine. It cannot cause disease because there is no whole germ. Hepatitis B vaccine made in yeast is the classic example. Such vaccines are safe and can be made in large amounts.
Other uses in medicine: molecular diagnosis. PCR finds very small amounts of virus or bacteria (like HIV in suspected AIDS patients) before symptoms show. ELISA detects antigens or antibodies. A probe (a labelled single-strand DNA or RNA) sticks to a matching sequence and shows where a faulty gene is.
Stem cells
A stem cell is a cell that is not yet specialised. It has two powers: it can renew itself by dividing, and it can differentiate (turn into) special cells. Sources:
- Embryonic stem cells: from a very early embryo; can become almost any cell (pluripotent).
- Adult stem cells: e.g. in bone marrow; make a smaller range, like all blood cells.
- Umbilical cord blood: collected at birth and stored in cord-blood banks.
Uses: bone-marrow transplant for blood cancers and thalassaemia; research to repair heart, nerve and skin damage. Concerns: use of embryos raises ethical questions; the body may reject donor cells; cells growing out of control could form tumours.
Gene therapy
Gene therapy means correcting a disease caused by a faulty gene by putting a working gene into the patient's cells or embryo. The first clinical use was in 1990 on a 4-year-old girl with ADA (adenosine deaminase) deficiency. Without ADA the immune system cannot work (a form of SCID).
- Take lymphocytes (white blood cells) from the patient's blood and grow them outside the body.
- Put a working ADA gene into them using a retrovirus vector (a virus made harmless).
- Return the cells to the patient.
Lymphocytes do not live forever, so the patient needs repeat doses. If the ADA gene is put into cells at an early embryo stage (or into bone marrow stem cells), it could be a permanent cure. Other treatments like bone-marrow transplant and enzyme injections also exist but are not a full cure.
GM organisms, Bt crops and RNAi
A genetically modified organism (GMO) is a plant, animal or microbe whose genes have been changed by genetic engineering. GM plants can: resist drought, cold, salt and heat; reduce the need for chemical pesticides; reduce loss after harvest; use minerals better; and have better food value, e.g. Golden rice rich in vitamin A.
Bt cotton: how the toxin works
The soil bacterium Bacillus thuringiensis (Bt) makes Cry proteins that kill certain insects: lepidopterans (bollworms, tobacco budworm), coleopterans (beetles) and dipterans (flies, mosquitoes). The bacterium keeps it as an inactive protoxin crystal, so it does not harm itself. In the insect gut, the alkaline pH dissolves the crystal and the protein becomes active. It binds to gut cells, makes pores, the cells swell and burst, and the insect dies. The genes are called cry: cryIAc and cryIIAb control cotton bollworms; cryIAb controls corn borer. Humans and cattle have acidic stomachs and lack the matching receptors, so this toxin does not harm them.
Pest-resistant plants by RNAi
The nematode Meloidogyne incognita infects tobacco roots. Scientists added nematode genes to the tobacco plant (using Agrobacterium) so the plant made both sense and anti-sense RNA. These pair up into double-stranded RNA, which starts RNA interference (RNAi): the matching mRNA of the nematode is silenced, so the parasite cannot survive in the root. RNAi is a natural defence found in all eukaryotes.
Transgenic animals
An animal whose DNA has been changed to carry and express a foreign gene is transgenic. Rats, rabbits, pigs, sheep, cows and fish have been made; over 95% are mice. Why make them?
- Normal physiology and development: to study how genes control growth, e.g. insulin-like growth factor.
- Study of disease: models for cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer's.
- Biological products: e.g. α-1-antitrypsin for emphysema. In 1997 Rosie, the first transgenic cow, gave milk with human α-lactalbumin (2.4 g per litre), better for human babies than normal cow milk.
- Vaccine safety: polio vaccine tested on transgenic mice before use on monkeys.
- Chemical safety (toxicity testing): animals with genes that make them more sensitive give results faster.
Biosafety, biopiracy and patents
Ethics and biosafety: changing living things can affect the environment and health in ways we cannot fully predict. India set up the GEAC (Genetic Engineering Appraisal Committee) to judge whether GM research is valid and whether releasing GM organisms for public use is safe.
Patent: a legal right given by a government to an inventor, so others cannot copy the invention for a fixed time. Problems arise when companies try to patent products based on traditional knowledge or on a country's biological resources.
Biopiracy: using bio-resources or traditional knowledge of a country without permission and without fair payment. Examples: an American company got a patent (1997) on a basmati-like rice made by crossing Indian basmati with semi-dwarf varieties; attempts were also made to patent uses of turmeric and neem. India challenged these, and in 2002 amended its Patents Bill to cover such issues. Rich nations have money and technology; poorer nations like India have biodiversity and traditional knowledge. Fair benefit-sharing is needed.
Try it: GM or not?
Next time you see a cotton shirt, remember that the cotton was probably Bt cotton. Make a two-column list at home: "Benefits of GM crops" and "Worries about GM crops". Add at least four points each (e.g. fewer sprays vs. pests becoming resistant). Then use the 3D menu to see each application and add the one you think is most useful.
Board exam tip: frequent questions are: how Bt toxin kills insects and why it does not harm the plant; steps of insulin production by Eli Lilly; RNAi in tobacco; first gene therapy; uses of transgenic animals; define biopiracy with an Indian example; role of GEAC.
Key formulas and definitions
- Insulin = chain A (21 amino acids) + chain B (30) joined by disulfide bonds; pro-insulin also has a C-peptide
- Recombinant vaccine = only the antigen protein, made in yeast/bacteria (e.g. Hepatitis B)
- Stem cell = self-renewal + differentiation
- Gene therapy (ADA, 1990): lymphocytes out → ADA gene via retrovirus → cells back
- Bt: cry gene → protoxin → alkaline gut → active toxin → pores → death (cryIAc, cryIIAb cotton; cryIAb corn)
- RNAi: dsRNA silences matching mRNA (tobacco vs Meloidogyne incognita)
- GEAC = Genetic Engineering Appraisal Committee
- Biopiracy = using bio-resources/traditional knowledge without permission or fair payment
Worked examples
1. Why does the Bt toxin not kill the Bacillus bacterium or the cotton plant?
It is stored as an inactive protoxin. It becomes active only in the alkaline gut of the insect. The plant and bacterium do not have that alkaline gut or the receptors.
2. How was the C-peptide problem avoided in making human insulin?
Chains A and B were made separately in E. coli from separate DNA pieces and then joined by disulfide bonds. Pro-insulin was never made, so no C-peptide had to be cut out.
3. Why does a patient given ADA gene therapy through lymphocytes need repeat treatment?
Lymphocytes have a limited life. When they die, the added gene is lost, so new engineered cells must be given again.
4. A recombinant Hepatitis B vaccine contains no virus. How does it still protect?
It has the virus surface protein (antigen). The immune system learns to recognise this protein and makes memory cells, which fight the real virus later.
5. How does RNAi make tobacco resistant to nematodes?
Genes of the nematode are put into the plant to make sense and anti-sense RNA. These form double-stranded RNA, which silences the matching nematode mRNA. The nematode cannot survive in the root.
6. A company patents a traditional Indian use of turmeric for wound healing. Is this biopiracy? Why?
Yes. It uses traditional knowledge that already existed in India without permission or fair benefit to the people who developed it.
Common mistakes
- Saying the Bt toxin is active in the plant. It is an inactive protoxin until the alkaline insect gut activates it.
- Writing that humulin was made from pro-insulin in E. coli. Chains A and B were made separately and joined.
- Thinking gene therapy with lymphocytes is a permanent cure. Only an early-stage (embryo or stem cell) insertion can be permanent.
- Mixing up biopiracy and patent. A patent is a legal right; biopiracy is unfair use of resources or knowledge without permission.