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Genetic Engineering
Searching for a target for the treatment
of atherosclerosis
RISK FACTORS
Consumption of foods high in saturated fats and a sedentary lifestyle increase
the risk of suffering from cardiovascular diseases such as atherosclerosis.
WHAT IS ATHEROSCLEROSIS?
Atherosclerosis is a vascular disease caused by the accumulation of fats on
the walls of the blood vessels. There are many different signs and degrees of
severity.
Normal artery
Moderate
atherosclerosis
Severe atherosclerosis
Formation of atheromatous plaque, which obstructs the blood flow.
“BAD” OR LDL CHOLESTEROL
• When
there is too much
cholesterol in the diet, it either
ends up being stored as fat or
continues circulating in the blood
in the form of LDL (low-density
lipoprotein) or what is more
commonly known as “bad
cholesterol”.
• LDL is one of the components of
the atheromatous plaque.
ACCORDING TO THE WORLD HEALTH ORGANISATION
(WHO)
•Although heart attacks and strokes are major killers in all parts of the world,
80% of premature deaths from these causes could be avoided by controlling
the main risk factors
•Every year, an estimated 17 million people globally die of cardiovascular
diseases (CVD), particularly heart attacks and strokes.
•CVDs occur almost equally in men and women.
•Are the leading cause of death in developing countries, as well as developed
ones.
THE MACROPHAGES, A “CLEAN-UP SYSTEM”
• The macrophages act as a “cleaning” system to stop the cholesterol from
being deposited on the walls.
• This clean-up system is efficient if the increased cholesterol is not too
excessive.
LDL
Macrophage
Oxidised LDL
Oxidation of LDL
HOW DOES AN ATHEROMATOUS PLAQUE FORM?
Proliferation
of endothelial
cells
If the quantities of cholesterol are very
excessive:
Immune
system
activation
The macrophages continue to pick up the LDL
Foam
cell
But, once they have engulfed large amounts, they turn
into what is known as “foam” cells.
LDL
Macrophage
These induce inflammation and the proliferation of
cells in the artery wall
Oxidation of LDL
Formation of the atheromatous plaque
Oxidised LDL
RESEARCH INTO ATHEROSCLEROSIS
One of the objectives is:
To understand how macrophages are involved in the regulation of cholesterol levels and what role they
play in the development of atherosclerosis.
?
STUDY OF THE LDL RECEPTOR AND MYLIP
We know that the macrophages recognise oxidised LDL thanks to some
receptors.
We know that a protein in the macrophages called MYLIP breaks down the LDL
receptor.
Macrophage
LDL receptor
Oxidised LDL
Oxidation of LDL
If the macrophages produce MYLIP in
large quantities, they ingest less
cholesterol.
MYLIP, A POTENTIAL THERAPEUTIC TARGET
Scientists are studying the MYLIP protein in depth because they think that it
could be regulated by a drug and thus stop the macrophages from ingesting
less cholesterol.
Macrophage
Therapeutic target
Oxidised LDL
Oxidation of LDL
HOW CAN WE STUDY THE PROTEINS?
• Large quantities of a protein are needed
to study its function.
•One of the tools in molecular biology
which allows us to study the proteins is
genetic engineering.
•Genetic engineering is the technology of
the manipulation and transfer of DNA
from one organism to another.
FIRST WE CLONE THE GENE OF THE PROTEIN OF INTEREST:
MYLIP
Human cell
ADN
MYLIP gene
We insert it into a fragment of
circular DNA called plasmid
WE PERFORM A BACTERIAL TRANSFORMATION WITH THE
GENE OF THE PROTEIN OF INTEREST: MYLIP
1. Bacterial
2. Selection of
transformation
transformed bacteria
3. Bacterial growth
THE GENE OF THE MYLIP PROTEIN IS THEN INTRODUCED
INTO CELLS IN ORDER TO PRODUCE MORE
Protein
MYLIP
3. Bacterial
4. We isolate
5. Production
growth
the DNA
of the protein
(introducing the DNA
into eukaryotic cells)
HOW DO WE DO A BACTERIAL TRANSFORMATION?
?
With drastic changes in
temperature and by adding
cations, we help the entry
of the DNA in the form of
plasmid into the cells.
HOW CAN WE MAKE SURE THAT TRANSFORMED BACTERIA
WILL GROW?
?
The plasmid has a gene
that is resistant to an
antibiotic.
HOW CAN WE MAKE SURE THAT TRANSFORMED BACTERIA
WILL GROW?
HOW DO WE ISOLATE THE PLASMID DNA FROM THE
BACTERIAL CULTURE?
?
Doing a Mini-prep:
Using various solvents and centrifugation cycles, we gradually discard the
different components
WITH THE DNA ISOLATED, WE CAN GET ON WITH THE
RESEARCH!
5. Production of the protein: (introducing the DNA
into eukaryotic cells).
6. Once the scientists obtain
the protein, they can then:
• Study its role in cholesterol regulation
• Search for new drugs for atherosclerosis.
Put on a lab coat!!
You too can be a researcher!!
Researchers who have contributed to the writing of this presentation: Theresa León,
Jonathan Matalonga, Barcelona University
AUTHOR
FUNDED BY:
PROJECT PARTNERS:
This work is under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported licence. To see a copy
of this licence, visit http://creativecommons.org/licenses/by-nc-nd/3.0/