A-level Gene technology. No copyright infringement intended.

1. 12a Bilegdemberel

2. Gene technology is the manipulation of
genes in living organisms. Genes from one
organism may be inserted into another. This
may be done within the same species ( for
example in gene therapy ) or genes may be
transferred from one species to another.
What is gene technology

3.  Insulin is a small protein. It is hormone
secreted by β cells in the islets of Langerhans
in the pancreas in response to raised blood
glucose concentration. In type I one diabetes,
no or insufficient insulin is secreted, and the
person has to inject insulin. This used to be
obtained from animals such as pigs. Today,
almost all insulin used in this way is obtained
from genetically modified bacteria.
Gene technology for insulin production

4.  The amino acid sequence of insulin was
already known. From this, the probable base
sequence of the gene that codes for it, and of
the mRNA transcribed from the gene could be
worked out.
Identifying the insulin gene

5.  Messenger RNA was extracted from β cells.
These cells express the gene for insulin, so
much of this mRNA had been transcribed from
this gene. The appropriate mRNA was then
incubated with the enzyme reverse
transcriptase, which built single-stranded
cDNA molecules against it. These were then
converted to double-stranded DNA- the insulin
gene.
Making the human insulin gene

6.  Some extra single-stranded DNA was then
added to each end of the DNA molecules.
These are called sticky ends. Because they are
single stranded, they are able to form
hydrogen bonds with other single-stranded
DNA, enabling DNA molecules to join up with
one another. This is important in a later stage
of the process.
Making the human insulin gene

7.  Multiple copies of the DNA were then made
using DNA polymerase. This can be done using
the polymerase chain reaction, or PCR. A small
amount of DNA is incubated with DNA
polymerase in a repeated sequence of
changing temperatures, enabling a huge
number of copies to be made in a relatively
short period of time.
Cloning the DNA

8.  A plasmid is a small, circular DNA molecule found
in many bacteria. A plasmid was cut open using a
restriction enzyme.The restriction enzymes make
a stepped cut across the DNA molecule, leaving
single stranded regions.
 The cut plasmid and the insulin gene were then
mixed together, along with the enzyme DNA
ligase. Complementary base pairing took place
between the sticky ends of the cut plasmids. DNA
ligase then joined up the sugar-phosphate
Inserting the DNA into a plasmid vector

9.  Backbones of the DNA strands. This resulted in
closed plasmids containing the insulin gene.
 Genes conferring resistance to an antibiotic
were also introduced into the plasmids, next to
the insulin gene.
 Not all of the plasmids took up the gene. Some
just closed back up again without it.
Inserting the DNA into a plasmid vector

10.  The plasmids were mixed with a culture of the
bacterium Escherichia coli. About 1% of them
took up the plasmids containing the insulin
gene.
Inserting the plasmid vector into a
bacterium

11.  Antibiotics were then added to the culture of
E. Coli bacteria. The only ones that survived
were the ones that had succcessfully taken up
the plasmids containing the antibiotic
resistance gene. Most of these plasmids would
also have contained the insulin gene. Most of
the surviving E. Coli bacteria were therefore
ones that now contained the human insulin
gene.
Identifying the genetically modified
bacteria

12.  The bacteria were then grown in fermenters,
where they were provided with nutrients and
oxygen to allow them to form large
populations. Reproduction is asexual, so all the
bacteria were genetically identical.
 This is now done on a large scale. The bacteria
synthesise and secrete insulin, which is
harvested from the fermenters and purified
before sale.
Cloning the bacteria and harvesting the
insulin

13.  It is identical to human insulin, because it is
made following the genetic code on the
human insulin gene. Insulin obtained from the
pancreas of an animal is slightly different, and
therefore may have different effects when
used to treat diabetes in humans.
 Large quantities of insulin can be made
continuously using E. Coli, and this can be done
under controlled conditions.
Advantages of insulin produced by
gene technology

14. Only small quantities of insulin can be obtained
from the pancreas of an animal, and it is not
easy to purify the insulin to produce standard
product that is safe for medicinal use.
Many religions and cultures, and also many
individuals, are uncomfortable with idea of
harvesting insulin from a dead animal for use in
humans.
Advantages of insulin produced by
gene technology

15.  In bacteria, each gene is associated with a
region of DNA called a promoter. The enzyme
RNA polymerase must bind to the promoter
before it can begin transcribing DNA to
produce mRNA.
 It is therefore important to ensure that there is
a promoter associated with the human insulin
gene when it is inserted into E.Coli
Promoters

16. The antibiotic resistance genes added to the
plasmids along with the human insulin gene act
as markers. They make it possible to identify the
bacteria that have taken up the gene.
There is a concern that using antibiotic resistance
genes as markers could increase the likelihood of
the development of populations of harmful
bacteria that are resistant to antibiotics. Today,
most common markers used are genes that code
for the production of fluorescent green protein.
The gene for this protein can be inserted along
with the desired gene. Cells that fluoresce green
are therefore likely to have taken up the desired
gene.
Markers

17. Electrophoresis is way of separating strands of
DNA of different lenghts.
 Cut DNA by restriction enzymes
 Place on agarose gel
 Apply current
 Fragments travel toward anode
 Short fragments travel further
 Visualise DNA with UV light
Electrophoresis

18. Cystic Fibrosis (CF) is a genetic condition resulting
from a mutation in a gene that codes for a
transporter protein called CFTR. CFTR protein
actively transport Cl- ions out of cells. When CFTR
protein is not working, high concentration of
chloride ion builds up inside the cell. It is because
chloride ions are actively transported into the cell
but not out of the cell. Therefore water moves
into the cell resulting in thick and sticky mucus.
Cystic Fibrosis

19.  The abnormality thick mucus collects in the
lungs, interfering with gas exchange and
increasing the chance of bacterial infections.
 The pancreatic duct may also become blocked
with sticky mucus, interfering with digestion in
the small intestine.
 Reproductive passages , such as vas deferens,
may become blocked, making a person sterile.
Cystic Fibrosis

20. Cystic fibrosis

21.  Finding out the genes that a person has is called
genetic screening. Genetic screening can be used:
 To identify people who are carriers, that is who
have a copy of a harmful recessive allele, such as
the cystic fibrosis in the family could therefore
find out if they are both hererozygous and
therefore might have a child with cystic fibrosiss.
 In pre implantation genetic diagnosis, to check the
genes of an embryo produced in vitro before it is
placed in the mother’s uterus.
Screening for genetic conditions

22.  For prenatal testing, that is checking the genes
of an embryo or fetus in the uterus;
 To identify people who will develop a genetic
condition later in life
 To identify people with alleles that put them at
risk of developing other diseases.
Screening for genetic conditions

23. Thank you !