BiologyCBSE Class 12 · Biotechnology and its Applications

Gene Therapy and Genetically Engineered Insulin

Updated 15 October 2026 · Fact-checked

In 1983 Eli Lilly made human insulin by putting DNA for the A and B chains into E. coli plasmids, producing the chains separately and joining them by disulphide bonds. Gene therapy corrects a genetic defect with a normal gene; the first case (1990) treated ADA deficiency by adding ADA cDNA to the patient's lymphocytes via a retroviral vector.

Understand Gene Therapy and Genetically Engineered Insulin

Biotechnology in medicine. rDNA technology allows mass production of safe and more effective drugs. Recombinant therapeutics do not induce unwanted immune responses, unlike similar products from non-human sources. About 30 recombinant therapeutics are approved worldwide; 12 are marketed in India.

Genetically engineered insulin.

Adult-onset diabetes can be managed by taking insulin at regular intervals. Earlier, insulin was extracted from the pancreas of slaughtered cattle and pigs; some patients developed allergy or other reactions to this foreign protein.
Insulin has two short polypeptide chains, A and B, linked by disulphide bridges.
In humans it is made as a pro-hormone (proinsulin) with an extra stretch, the C peptide, which is removed during maturation; mature insulin lacks it.
The main challenge in rDNA production was getting insulin assembled into its mature form.
In 1983, the American company Eli Lilly prepared two DNA sequences corresponding to the A and B chains of human insulin and introduced them into plasmids of *E. coli*. The A and B chains were produced separately, extracted, and combined by creating disulphide bonds to form human insulin.

This avoids the C-peptide processing problem and gives human insulin without animal-protein allergy.

Flow chart (insulin): DNA for chain A → plasmid → E. coli → chain A; DNA for chain B → plasmid → E. coli → chain B → extract both → join by disulphide bonds → human insulin.

Gene therapy. A collection of methods to correct a gene defect diagnosed in a child or embryo: a normal gene is delivered into the individual or embryo so that it does the job the faulty (non-functional) gene cannot do.

First clinical gene therapy: 1990, to a 4-year-old girl with adenosine deaminase (ADA) deficiency. ADA is crucial for the immune system; the disorder is caused by deletion of the gene for ADA.
Existing treatments: bone marrow transplantation (some children) and enzyme replacement therapy (functional ADA injected). Neither is completely curative.
Gene therapy steps: (1) lymphocytes from the patient's blood are grown in culture outside the body; (2) a functional ADA cDNA is introduced into them using a retroviral vector; (3) the cells are returned to the patient.
Because these lymphocytes are not immortal, the patient needs periodic infusions of engineered lymphocytes.
If the ADA gene isolated from marrow cells were introduced into cells at early embryonic stages, it could be a permanent cure.

Flow chart (ADA): patient's lymphocytes → culture → add ADA cDNA via retrovirus → engineered lymphocytes make ADA → infused back → immune function improves → repeat periodically.

Molecular diagnosis (early detection). Conventional serum and urine tests cannot detect disease early. By the time symptoms appear, the pathogen is already numerous.

PCR amplifies nucleic acid of a pathogen present in very low concentration, before symptoms: used routinely to detect HIV in suspected AIDS patients, and to find mutations in genes of suspected cancer patients and other genetic disorders.
Probe and autoradiography: a single-stranded DNA or RNA tagged with a radioactive molecule (probe) hybridises to its complementary DNA in a clone of cells and is detected by autoradiography. A clone with a mutated gene does not appear on the film because the probe is not complementary to it.
ELISA works on antigen-antibody interaction: infection is detected by the presence of antigens (proteins, glycoproteins) of the pathogen or of antibodies made against it.

Draw and label: maturation of insulin. Checklist: a single chain folded as proinsulin with three segments; the middle segment (C peptide) cut away; the remaining two chains held by two S-S bridges. Labels: proinsulin, A chain, B chain, C peptide (removed), disulphide bonds, mature insulin.

Gene Therapy and Genetically Engineered Insulin: key facts

Insulin structure: $insulin = A chain + B chain (S-S bridges); proinsulin = A + B + C peptide$; C peptide is removed during maturation.
Eli Lilly method (1983): $A-DNA, B-DNA \to E . co l i plasmids \to separate chains \to join by S-S$; Avoids proinsulin processing.
ADA gene therapy (1990): $lymphocytes + ADA cDNA (retroviral vector) \to reinfused$; Periodic infusions needed; embryonic gene transfer could be permanent.
Molecular diagnosis: $P C R (low pathogen DNA, mutations); E L I S A (antigen-antibody); probe + autoradiography$; PCR detects HIV in suspected AIDS patients.
Recombinant therapeutics: $\approx 30 approved worldwide; 12 marketed in India$; No unwanted immune reactions.

How to answer Gene Therapy and Genetically Engineered Insulin questions

Questions ask about insulin structure and production, the ADA gene therapy procedure, or how a disease is diagnosed early.

1For insulin, give the problem with animal insulin (allergy), the structure (A and B chains, disulphide bridges, C peptide in proinsulin) and Eli Lilly's 1983 method.
2Explain the challenge: assembling mature insulin, and how separate chain production solved it.
3For gene therapy, define it, then give the ADA case: cause (gene deletion), older treatments and their limits, the three steps, and why infusions are repeated.
4Say what would make the cure permanent (gene transfer into early embryonic cells).
5For diagnosis, match technique to need: PCR (tiny amounts of DNA, mutations), probe with autoradiography (mutated gene does not light up), ELISA (antigen or antibody).
6Close with an advantage: early detection, no immune reaction to recombinant drugs.

Gene Therapy and Genetically Engineered Insulin in real life

The idea, met in an everyday situation. Answer the question in your own words, then compare with the model answer. The one-line hook is what to remember.

Choosing a diagnosis method

Suppose a blood bank must screen donated blood for HIV. A donor was infected only a few days ago and has no symptoms and very few virus particles, while another donor was infected a year ago.

Which technique will detect the early infection and why? Which technique can confirm the older infection, and on what principle?

Try it first, then show the solutionSolution

Early infection: very little viral nucleic acid; PCR amplifies it many times so it can be detected before symptoms appear.
Older infection: the body has made antibodies against HIV (and viral antigens are present); ELISA detects these by antigen-antibody interaction.
Conventional tests depending on symptoms would miss the early donor, so molecular methods make blood safer.

Answer: PCR for the early, low-level infection (amplifies viral nucleic acid); ELISA for the older infection (detects antibodies/antigens).

Memory hook: Too few viruses? Copy them (PCR). Antibodies made? Catch them (ELISA).

Tricks to remember Gene Therapy and Genetically Engineered Insulin

Short memory aids. Say each one aloud once, then use it on a question the same day.

A-B, no C
Mature insulin = A + B chains; the C peptide is cut out.
Use it when: Insulin structure.
1983 insulin, 1990 gene therapy
Insulin first (83), gene therapy later (90).
Use it when: Dates.
ADA = Always Daily (periodic) Additions
ADA gene therapy needs periodic infusions because lymphocytes are not immortal.
Use it when: Limitation of ADA gene therapy.

Gene Therapy and Genetically Engineered Insulin: solved examples

Ordered from board level to HOTS. Try each one before you open the solution.

Example 1: Why recombinant insulin · 2 marks · Very short answerBoard level

Why was insulin from cattle and pigs replaced by genetically engineered human insulin?

Try it first, then show the solutionSolution

Animal insulin from slaughtered cattle and pigs caused allergy or other reactions in some patients because it is a foreign protein (1 mark).
Recombinant human insulin is identical to the body's own insulin, can be mass-produced and does not cause unwanted immune responses (1 mark).

Answer: Animal insulin caused allergies; recombinant human insulin is safe and mass-produced.

Example 2: Eli Lilly's method · 3 marks · Short answerBoard level

Explain how Eli Lilly produced human insulin by rDNA technology. Why was the C peptide a problem?

Try it first, then show the solutionSolution

Insulin is made in humans as proinsulin with an extra C peptide, which must be removed to form mature insulin of A and B chains linked by disulphide bridges; assembling mature insulin was the main challenge (1 mark).
In 1983 Eli Lilly prepared two DNA sequences for the A and B chains and introduced them into E. coli plasmids (1 mark).
The A and B chains were produced separately, extracted and combined by forming disulphide bonds to give human insulin (1 mark).

Answer: Separate A and B chain genes in E. coli; chains joined by disulphide bonds, bypassing proinsulin processing.

Example 3: ADA gene therapy · 5 marks · Long answerHarder board level

Describe the first clinical gene therapy. Why is it not a permanent cure, and how could it become one?

Try it first, then show the solutionSolution

In 1990 a 4-year-old girl with adenosine deaminase (ADA) deficiency, caused by deletion of the ADA gene, was treated; ADA is crucial for the immune system (1 mark).
Bone marrow transplantation and enzyme replacement therapy (injected ADA) are not completely curative (1 mark).
Lymphocytes from the patient's blood were grown in culture; functional ADA cDNA was introduced using a retroviral vector; the cells were returned to the patient (1 mark).
These lymphocytes are not immortal, so periodic infusions of engineered lymphocytes are needed (1 mark).
A permanent cure could come from introducing the ADA gene isolated from marrow cells into cells at early embryonic stages (1 mark).

Answer: Lymphocytes + ADA cDNA (retrovirus) reinfused; repeated because cells die; embryonic gene transfer could cure permanently.

Example 4: Probe and autoradiography · 2 marks · Very short answerHarder board level

How can a radioactive probe identify a clone with a mutated gene?

Try it first, then show the solutionSolution

A probe (single-stranded DNA or RNA labelled with a radioactive molecule) pairs with its complementary DNA in a clone of cells, and the bound probe is detected by autoradiography (1 mark).
A clone with the normal gene hybridises and shows on the film; a clone with a mutated gene does not hybridise and does not appear (1 mark).

Answer: Normal clones bind the probe and show on film; mutated clones do not appear.

Example 5: Why lymphocytes? · 3 marks · Short answerHOTS / competency

Suppose a student asks why doctors put the ADA gene into lymphocytes rather than into, say, skin cells. Explain, and say what would happen if the retroviral vector were not used.

Try it first, then show the solutionSolution

ADA is crucial for the immune system, and lymphocytes are immune cells; correcting them restores immune function where it is needed (1 mark).
Lymphocytes are easily taken from blood, grown in culture and returned by infusion (1 mark).
Without a vector, the hydrophilic cDNA would not efficiently enter the cells; the disarmed retrovirus delivers the gene into the lymphocytes (1 mark).

Answer: Lymphocytes are the affected immune cells and easy to culture and return; the retroviral vector is needed to deliver the gene.

Example 6: Early diagnosis · 3 marks · Short answerHarder board level

Why are conventional methods of diagnosis unable to detect infections early, and how do PCR and ELISA overcome this?

Try it first, then show the solutionSolution

Conventional serum and urine tests and symptom-based diagnosis detect a pathogen only after it has multiplied to high concentration (1 mark).
PCR amplifies even a very low concentration of bacterial or viral nucleic acid, so infection (e.g. HIV) can be detected before symptoms (1 mark).
ELISA detects antigens of the pathogen or antibodies made against it by antigen-antibody interaction (1 mark).

Answer: Conventional tests need high pathogen load; PCR amplifies traces; ELISA detects antigens or antibodies.

Where students lose marks in Gene Therapy and Genetically Engineered Insulin

The slips examiners see most often, and what to write instead.

Mistake: Including the C peptide in mature insulin
Proinsulin and insulin are confused.
Wrong working
Insulin has three chains: A, B and C.
Right working
Mature insulin has A and B chains; C peptide is removed.
Do this: A-B, no C.
Usually costs: 1 mark.
Mistake: Wrong cells in ADA therapy
Students write bone marrow or RBCs.
Wrong working
ADA gene was inserted into red blood cells.
Right working
Into lymphocytes cultured from the patient's blood, using a retroviral vector.
Do this: Lymphocytes + retrovirus.
Usually costs: 1 mark.
Mistake: Calling ADA gene therapy a permanent cure
Overstatement.
Wrong working
Gene therapy permanently cured ADA deficiency.
Right working
Periodic infusions are needed; only early embryonic gene transfer could be permanent.
Do this: Mention 'not immortal' lymphocytes.
Usually costs: 1 mark.
Mistake: Insulin chains produced together
Students assume one gene.
Wrong working
A single gene for insulin was put into E. coli.
Right working
Two DNA sequences (A and B chains) in plasmids; chains made separately, then joined.
Do this: Two genes, two chains, one bond step.
Usually costs: 1 mark.

Exam tips

Quote the key facts: Eli Lilly 1983; A and B chains; disulphide bonds; C peptide removed.
For gene therapy, give the 1990 case details: 4-year-old girl, ADA deficiency, lymphocytes, retroviral vector, periodic infusion.
Name all three diagnostic tools and their uses.
Mention that recombinant therapeutics avoid unwanted immune responses.
Keep stem cell technology and vaccines in mind as other syllabus applications (hepatitis B vaccine from yeast).

Practice questions on Gene Therapy and Genetically Engineered Insulin

Write each answer on paper first. Open the hint only if you are stuck, and the answer only when you have finished.

Q1 · 1 mark · MCQBoard level
In mature human insulin, the A and B chains are held together by:
(a) peptide bonds (b) hydrogen bonds only (c) disulphide bridges (d) the C peptide
Hint
S-S.
Answer
(c) disulphide bridges.
Q2 · 1 mark · Assertion-reasonHarder board level
Assertion (A): A child given ADA gene therapy needs periodic infusions of genetically engineered lymphocytes.
Reason (R): The engineered lymphocytes are not immortal.
(a) Both A and R are true and R is the correct explanation of A.
(b) Both A and R are true but R is not the correct explanation of A.
(c) A is true but R is false.
(d) A is false but R is true.
Hint
Cells die.
Answer
(a). The corrected lymphocytes eventually die, so fresh engineered cells must be infused from time to time; only gene transfer at an early embryonic stage could be permanent.
Q3 · 2 marks · Very short answerHarder board level
What is enzyme replacement therapy for ADA deficiency, and why is gene therapy considered better?
Hint
Injected enzyme vs gene.
Answer
Functional ADA enzyme is injected into the patient; it is not curative and must be repeated. Gene therapy gives the patient's own cells a working ADA gene, so they make the enzyme themselves; introduced at an early embryonic stage it could be a permanent cure.
Q4 · 3 marks · Short answerHOTS / competency
Suppose E. coli were given a single gene coding for whole human proinsulin. Predict a problem with obtaining active insulin, and explain how Eli Lilly's approach avoided it.
Hint
Who removes the C peptide?
Answer
Proinsulin must be processed: the C peptide removed and the A and B chains correctly linked by disulphide bonds. Bacteria may not process human proinsulin correctly, so the product may not become mature, active insulin. Eli Lilly expressed the A and B chains separately from two DNA sequences, extracted them and joined them chemically by disulphide bonds, so no C peptide processing was needed.
Q5 · 4 marks · Case-basedHarder board level
Read and answer.
Suppose a hospital report reads: 'Patient, 4 years: recurrent infections; ADA gene deleted. Plan: harvest lymphocytes, culture, transduce with functional gene using a viral carrier, reinfuse; repeat every few months.'
(a) What does ADA stand for and why are infections recurrent? (1)
(b) What is the 'viral carrier'? (1)
(c) Why must the treatment be repeated, and what kind of gene transfer could avoid repetition? (2)
Hint
Retrovirus; immortal cells.
Answer
(a) Adenosine deaminase; it is crucial for the immune system, so its absence weakens immunity and infections recur. (b) A (disarmed) retroviral vector carrying functional ADA cDNA. (c) The engineered lymphocytes are not immortal, so they die and must be replaced periodically. Introducing the ADA gene (from marrow cells) into cells at an early embryonic stage could give a permanent cure.
Q6 · 2 marks · Very short answerBoard level
Name the techniques used for early molecular diagnosis and state the principle of ELISA.
Hint
Three techniques.
Answer
Recombinant DNA technology (probes), PCR and ELISA. ELISA works on antigen-antibody interaction: infection is detected by antigens of the pathogen or by antibodies made against it.

Test your recall

Answer in your head or on paper first, then open the card. Pulling an answer out of memory makes it stick far better than re-reading.

1.Who produced human insulin by rDNA technology and when?Show answer
Eli Lilly, 1983.
2.What is removed from proinsulin during maturation?Show answer
The C peptide.
3.Which disorder was treated by the first gene therapy?Show answer
Adenosine deaminase (ADA) deficiency.
4.Which vector delivered the ADA cDNA?Show answer
A retroviral vector.
5.Which technique detects HIV in suspected AIDS patients early?Show answer
PCR.
6.On what principle does ELISA work?Show answer
Antigen-antibody interaction.

Gene Therapy and Genetically Engineered Insulin: frequently asked questions

How is genetically engineered insulin made?

DNA sequences for the A and B chains of human insulin are put into E. coli plasmids; the chains are produced separately, extracted and joined by disulphide bonds.

What is gene therapy?

A set of methods to correct a gene defect by delivering a normal gene into a person's cells or an embryo to take over the function of the faulty gene.

How was ADA deficiency treated by gene therapy?

The patient's lymphocytes were cultured, given functional ADA cDNA using a retroviral vector, and returned to the patient; infusions are repeated periodically.

Why is ADA gene therapy not permanent?

The engineered lymphocytes are not immortal; a permanent cure would need gene transfer into cells at an early embryonic stage.

How does PCR help in early diagnosis?

It amplifies very small amounts of pathogen nucleic acid, so infections like HIV can be detected before symptoms appear.