1.

0 GENE TECHNOLOGY
1.1 INSULIN PRODUCTION
Objectives

By the end of the subtopic learners should be able to:

1. Define gene technology

2. Outline the synthesis of human insulin by bacteria
3. Explain the advantages of treating diabetics with human insulin produced by gene
technology.

 Gene technology involves the artificial manipulation, modification, and recombination of

DNA or other nucleic acid molecules in order to modify the genetic makeup of organisms.
 DNA molecules from two or more sources are combined either within cells or in vitro.
 There are then inserted into host organisms in which they are able to propagate.
 The method creates new combinations of genes and is referred to as genetic engineering.
 Genetic engineering in bacteria can be broken into five stages (fig.1.1.1).

Fig.1.1.1: Stages of genetic engineering.

Some of the applications of genetic engineering include:

o Basic research on gene structure and function

o Production of useful proteins by novel methods for example insulin
o Generation of transgenic plants and animals.
o Medical diagnosis and treatment
o Genome analysis by DNA sequencing

Insulin production

 Insulin is a hormone that is produced and secreted by the beta cells of the pancreas' Islets
of Langerhans.
 It is a simple protein which consists of two polypeptides.
 The hormone has a total of 51 amino acids, 30 of which constitute one polypeptide chain,
and 21 of which comprise a second chain.
 Insulin is responsible for controlling blood glucose levels and its shortage causes a condition
referred to as diabetes.
 Bacteria are used as hosts in insulin production because of their ability to readily take up
plasmids and produce proteins carried by the plasmid.

Steps for insulin production

 Insulin can be produced using bacteria (E.coli) through Recombinant DNA technology.
 Recombinant DNA technology involves joining together DNA from different species.
 Several steps are involved in insulin production as outlined below.

1. Extraction
o The plasmid DNA is isolated from bacteria.
o A copy of the human insulin gene is obtained.
o This is done by isolating mRNA from the human pancreas.

2. Digestion by restriction enzymes

o Restriction enzymes are used to cut the plasmid DNA.
o Plasmid DNA contains specific sequences which are known as restriction sites to allow
cleavage of specific sites by restriction enzymes.
o Restriction enzymes cut DNA at specific points by recognising specific sequences on the
DNA molecule.
o They are naturally found in bacteria and they protect bacteria against viruses.
o Restriction enzymes can leave either open stick ends (length of unpaired bases) or blunt
ends.
 o The same restriction enzymes (restriction endonucleases) are also used to cut insulin
DNA.
o However, they do not digest their host DNA because bacterial DNA is methylated.

Fig.1.1.2: Restriction digestion of the plasmid and the insulin gene

3. Insertion
o The isolated insulin DNA is then inserted into a plasmid vector.
o The cut plasmids are mixed with the insulin gene and DNA ligase enzymes are added.
o DNA ligase enzymes are enzymes that are used to join together plasmid DNA with the
insulin DNA to form recombinant DNA molecule
 Fig.1.1.3: Diagrammatic representation of the insertion step.

4. Transformation
o The recombinant molecule is then inserted back into the bacterium which will act as a
host.
o The process whereby bacteria take up recombinant molecules is referred to as
transformation.
o The cells which have taken up the plasmid are called transformants.
o Heat shock is usually introduced to induce transformation.

5. Selection
o The transformed cells are identified and selected basing on marker genes.
o A marker gene is a gene that allows genetically modified cells to be readily selected.
o An example is the gene that encodes antibiotic resistance.
o Antibiotic resistance will act as a selectable marker.
o These selectable markers are found on plasmids.
o The bacterial host cell will divide and produce copies of the plasmid carrying the insulin
gene (fig 1.1.4).

6. Extraction and purification

o Insulin is then extracted from the bacterial culture.
o It is then purified.
Fig.1.1.4: Summary of the steps in the production of insulin

Advantages of using insulin to treat diabetes`

 Before bacteria was used to produce human insulin diabetes were injected with insulin
isolated from pigs or cattle.
 However, pig or cattle insulin is not identical to human insulin and there were cases of
immune response.
 There are minor differences in the amino acid composition between cow or pig insulin and
human insulin (fig.1.1.5).
 Fig.1.1.5: Differences between pig and human insulin.

 Insulin produced using bacteria is chemically similar to human insulin and there are less
chances of immune response.
 It can also be easily recognised by human insulin receptors on human cell surface
membranes hence duration of response is shorter.
 Insulin produced by gene technology eliminates the problems related to the development
of tolerance to cow or pig insulin.
 Ethical issues raised by use of cow or pig insulin are eliminated when insulin is produced
through gene technology.
 For instance, religious objections of pig insulin by Muslims and Christians or objections of
both pig and cattle insulin by vegetarians.
 Extraction of insulin from pancreas of cows or pigs is expensive as compared to production
of insulin through gene technology.
 Production of insulin through gene technology also eliminates problems related to animal
rights issues as there is no need to slaughter any animal.