Genes and Genetic Engineering - Applications of Gene Technology

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Dec 11, 2012 (4 years and 6 months ago)

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T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
1







11.4


Applications of Gene Technology



Gene technology has important applications in many areas including biotechnology, medicinal drug
production, gene therapy, agriculture and horticulture


Genetic engineering can generate many potential benefit
s for humans. But there are hazards too.


The economic advantages may be outnumbered by environmental and ethical drawbacks or dangers.

The issues require balanced and informed judgements


Principles of Genetic Engineering



The first step in genetic
engineering usually involves inserting a short piece of donor DNA

into the DNA of a recipient organism.


The recipient organism is unrelated to the donor e.g. from human to bacterium.


The recipient organism then acquires the ability to synthesise the
protein for which the donor DNA
codes.


The outcomes are new varieties of organisms


mostly microorganisms


Microoganisms are preferred because they are easier to modify


To remove the DNA from a cell the cell membrane needs to be disrupted and the nucl
eus broken
open. The method used depends on the type of cell.


One common way to disrupt a cell is a detergent called sodium dodecyl sulphate (SDS) which breaks
down cell membranes (including the nuclear membrane) and cell walls


The protein framework of

the chromosomes is removed by incubation with a protease


Once the DNA has been isolated from the rest of the cell, the part of the DNA molecule which
contains the required gene has to be cut out and the rest of the DNA discarded. (There are on
average 22
00 genes on each human chromosome)


It is important to know the base sequence of the gene required


so that either a gene probe can be
used and/or the appropriate restriction endonuclease selected


Genes are isolated using enzymes which cut across DNA mol
ecules at particular positions


RESTRICTION ENDONUCLEASE ENZYMES.


Several different types of restriction endonuclease exist


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
2

Restriction endonuclease enzymes are found naturally in bacteria where their function is to chop up
and destroy the DNA of any
viruses that infect the cell. They are so named because the RESTRICT
the multiplication of viruses


A given restriction endonuclease cuts a bacterial plasmid open at a specific site which is determined
by the base sequence in that region


The same enzyme
will cut donor DNA wherever an identical base sequence occurs


It is characteristic of most restriction endonucleases that they cut the 2 strands of DNA at slightly
different points


the result is that each end of the foreign DNA segment has a short row (
4 bases
long) of unpaired bases which match the complementary bases at each end of the opened up
plasmid. These are called STICKY ENDS


Genetically Engineered Microorganisms


The next stage is to insert the isolated gene into a VECTOR. A vector is a piec
e of DNA that can
take the gene into the chosen microbe.


A common vector is a small circular molecule of DNA called a PLASMID. These occur naturally in
bacteria in addition to the larger molecule of chromosomal DNA


Plasmids are easily isolated from a ba
cterium and they can be re
-
introduced into another bacteria
relatively easily too.


As mentioned above, the same restriction endonulclease is used to cut the plasmid DNA as was
used for to isolate the gene from the donor DNA. This means that the sticky en
ds produced will fit
together


The donor gene is then inserted into the plasmid loop using the enzyme LIGASE. Ligase catalyses
the LIGATION reaction which joins 2 sections of DNA together


the hydrogen bonds form between
complementary bases.


Ligase occu
rs naturally in nuclei where it ‘repairs’ DNA damaged in replication


The new DNA is called RECOMBINANT DNA


Once in position, the foreign DNA (donor gene) replicates along with the rest of the plasmid every
time the bacterial cell divides


Plasmids contai
ning the donor gene must now be transferred into the microbe


A culture of the intended bacterial recipients is placed in cold calcium chloride solution for 30 mins


This changes the membranes of the bacteria


making them more permeable to the plasmids


P
lasmids with the recombinant DNA are added to the culture medium and warmed up for a short
time.


Some of the bacteria take up plasmids


those that do are said to have undergone
TRANSFORMATION


This process is not very efficient


only a small proportio
n of the bacteria are transformed

T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
3



Genetic Markers


The transformed cells must now be identified and isolated.


One technique for doing this involves inserting a marker gene into the plasmids as well as the donor
gene


This marker gene may make the bacter
ia resistant to a particular antibiotic


If the culture containing the transformed bacteria is grown on a medium containing the antibiotic,
bacteria with the plasmids that have the antibiotic resistance marker gene (and the donor gene) will
survive and gro
w better than those without it


Replica plating can the be done


This eliminates colonies without the resistance gene (and therefore no donor gene)


The donor gene also has to be made to start working once it has been transferred to the recipient
bacteria


Not all genes in a bacterium are switched on all the time


It is possible to overcome this by using PROMOTER GENES that control the expression of the main
gene.


They indicate where transcription should begin and ensure mRNA is translated by the ribosomes


The promoter genes are inserted into the plasmid, with the donor gene and the marker gene before
transformation


Large Scale Culturing


Since the gene product is often needed in large amounts the transformed bacteria are cultured on a
large scale in an i
ndustrial fermenter


The medium in it contain all the nutrients the bacteria need for rapid growth and reproduction and
supplies the oxygen they need for respiration


In favourable conditions the bacteria will divide as often as every /2 hour


A huge clone

of the bacteria can be produced within days


As the bacteria grow, the gene within the plasmids synthesises its protein


The product is extracted from the fermenter for commercial use


Useful substances produced by genetic engineering include antibiotics,

hormones (insulin, human
growth hormone, factor VIII) and enzymes (those in biological washing powders, produced for use in
food industry)


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
4

Genetic engineering is also used to make pest resistant varieties of crops


genes producing
chemicals which confer

resistance to attack by fungi or insects can increase yields and reduce the
amount of pesticide which needs to be used


The Polymerase Chain Reaction (PCR)


PCR is like a nuclear chain reaction in that it proceeds at an ever


increasing rate


PCR can amp
lify tiny amount of DNA into quantities large enough for scientific analysis (e.g. at the
scene of a crime just a few white blood cells or a tiny sample of sperm may be found)


It is the basis of genetic fingerprinting / DNA fingerprinting / genetic profil
ing (all same thing!)


The uses of PCR include amplifying DNA from samples of tissues from extinct animals (e.g.
Tasmanian wolf) to establish their closest relatives and amplifying DNA from buried human bodies
where some soft tissue has been preserved. Th
is helps to understand the migration of early human
populations


PCR uses the enzyme DNA polymerase


this is the enzyme which occurs naturally in cells and
catalyses the replication of DNA in the nucleus


The first stage of PCR is to heat the DNA to (95
o
C



this makes the 2 polynucleotide strands
separate)


DNA nucleotides are added and the mixture is cooled to 40
o
C. The DNA polymerase attaches the
new nucleotides to each strand (as in normal replication)


The process can then be repeated endlessly


prod
ucing 2,4,16,32 DNA molecules and so on


Once a large enough sample has been generated using PCR it can be analysed and compared with
samples from known sources


This allows us to compare DNA from the same or different species.


Forensic scientists often h
ave to compare DNA found at the crime scene with DNA from suspects


In each case the first stage is to cut the DNA into short lengths with restriction enzymes


This produces DNA fragments of varying lengths


because the sequence of bases where the
enzymes

cut occur at irregular intervals along the DNA molecules


The lengths of these fragments will also be different in different individuals


since we all have a
unique sequence of nucleotides in our DNA. The same restriction endonuclease will chop up our
D
NA in different places


The DNA fragments are then separated using electrophoresis


The mixture of DNA fragments is placed at one end of a long piece of agar jelly in a trough containing
a dilute solution of ionic salts


Electrodes are placed in the soluti
on at either end and a voltage is applied


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
5

The phosphate groups in the fragments of DNA give them a negative charge


so they are attracted
through the gel towards the positive electrode. The smaller fragments move more rapidly than the
larger ones


so t
he different sized fragments are separated in a similar way to molecules in
chromatography


The pattern of fragments is like a ‘fingerprint’
-

this fingerprint is unique to each of us. The only
exception is identical twins


they share the same pattern


To be able to compare fingerprints they must be made visible.


The pattern of DNA fragments in the gel is transferred to a nitrocellulose sheet


This is done overnight and produces an ‘imprint’ of the pattern of the fragments from the original gel
onto

the nitrocellulose sheet


A probe labelled with radioactivity is then used to reveal the position of the bands on the sheet that
contain the DNA sequence you are interested in.


To be able to pick out a particular gene or genes you first need to know its
base sequence


The gene probe can then be made which will seek out the particular gene. The gene probe may be
labelled


often done by using a radioactive isotope of phosphorus (
32
P) as a component of the
phosphate groups


A general probe can be used whic
h make all the bands show up or a specific probe can be used. A
specific probe is a piece of single stranded DNA that is complementary to the base sequence of the
specific stretch of DNA


The nitrocellulose sheet is incubated with the probe in a sealed pl
astic bag containing a buffer
solution. The probes then bind (base pair) to the appropriate DNA fragments


The sheet is removed and placed next to an unexposed piece of photographic film


The radioactivity in the probe causes a band to show up on the film


Other examples of where the technique has been used:




Paternity cases




Relationships within families




Social behaviour in animals




Immigration cases


Gene Therapy


This is the field of medicine that aims to treat people with genetic disorders by giving t
hem a copy of
a healthy gene to overcome the problems produced by the mutated gene


This procedure is called GENE THERAPY


There are 2 types of cells in our bodies

T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
6


1.

germ line cells


give rise to eggs and sperm


2.

somatic cells


‘normal’ body cells


Geneti
c changes to somatic cells cannot be passed onto future generations. Changes to germ line
cells can be passed onto future generations


The genetic engineering of somatic cells (somatic gene therapy) is legal, but tightly regulated.


The genetic engineer
ing of germ
-
line cells (germ
-
line therapy) is not permitted in any country


Gene therapy is being developed to treat the genetic disease cystic fibrosis


Common in white North Americans and Northern Europeans


1 in 25 carriers

1 in 2000 sufferers


Most peo
ple have a gene that produces a protein called the CYSTIC FIBROSIS TRANSMEMBRANE
REGULATOR (CFTR)


This is a complex molecule made up of 1480 amino acids and it is one of the essential channel
proteins in cell membranes


The function of this particular pro
tein is to transport chloride ions through the cell membrane


Cause is a recessive mutation of chromosome 7


The gene on this chromosome codes for the CHLORIDE CHANNEL PROTEIN
-

CFTR


Cystic
Fibrosis Transmembrane Regulator)


It is located in the cell su
rface membranes and allows diffusion of chloride ions into and out of
epithelial cells


In CF sufferers this protein does not function because there are 2 copies of the faulty gene


Responsible gene was cloned in 1989


Usually the cause is a deletion muta
tion


3 base pairs are missing so codon 508 in the mRNA is
missing


As a result, the amino acid phenylalanine (F) is not inserted at position 508 in the 1480 aa protein


The mutation is ^F508 (^ = d for deletion)


There are at least 400 other mutations of

this protein but this is the most common


This mutation affects the 3D shape of the channel protein so that it can no longer transport chloride
ions through a membrane


Symptoms


Normal epithelial cells form mucus glands which secrete mucus


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
7

In CF patient
s this mucus is unusually thick and sticky


In CF the normal outward flow of Cl
-

ions from the cell is prevented


So, chloride ions build up, a negative charge builds up inside the cell so more sodium ions (+’ve)
move in to balance the

‘ve charge


The hig
h ion concentration in the cell prevents water leaving the cell and so mucus is abnormally
thick and sticky


The lungs, pancreas and liver are the most affected organs


Sweat produced is unusually salty


The mucus clogs up airways in the lungs, branches of

the pancreatic duct and bile duct from the liver
and so there are repeated lung infections and digestive problems


50% of sufferers die by age 20, but average life expectancy has now increased to about 20
-
30



Treatment


Vigorous physiotherapy to dislodge

the mucus from the lungs


Enzyme supplements to aid digestion

Antibiotics to fight infections (in respiratory and digestive
systems)


Heart lung transplants for severe cases


Clinical trials are underway for gene therapy, attempting to insert CFTR gene i
nto lung / tracheal
epithelial cells to replace the defective genes


2 techniques are being tested


1.

wrapping the CFTR genes in lipids that can be absorbed through the csm


2.

inserting CFTR genes into harmless viruses that are then allowed to ‘infect’ the cel
ls


1. wrapping the CFTR genes in lipids that can be absorbed through the csm


This method uses tiny lipid droplets called LIPOSOMES


These can fuse with the phospholipid molecules that make up the csm and so carry genes into the
target cells


CFTR genes a
re isolated from healthy human cells, cloned and inserted into plasmids using
recombinant DNA technology


The genes are inserted into liposomes


Aerosol sprays are used to get the liposomes into the lungs


like inhalers for asthma sufferers


In the lungs,

the liposomes fuse with the csm of epithelial cells and the genes are transported into the
cell

T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
8


One problem with the technique is developing a fine enough spray to get the liposome through the
narrow bronchioles in the lungs and into the alveoli


Also, o
nly a small proportion of the genes that are absorbed into cells are actually expressed


These are problems which must be overcome before this form of gene therapy becomes widely
available as a treatment for CF



2.

inserting CFTR genes into harmless viruses
that are then allowed to ‘infect’ the cells


In this method viruses called ADENOVIRUSES are used


These viruses reproduce themselves by injecting their DNA into host cells


The viral DNA uses the cell’s enzymes and ribosomes to replicate and produce copies



which then
reconstruct the rest of the virus before being released from the cell


Adenoviruses infect the cells of the airways in the lungs, and they are adapted for replication in the
epithelial cells


They normally cause colds and other respiratory di
seases


The adenoviruses used for gene therapy have been modified so they infect the cells but do not cause
the diseases


The modification is that the genes which allow the virus to replicate are disabled


The modified viruses are cultured in epithelial ce
lls grown in the laboratory and exposed to plasmids
that have the CFTR gene


The CFTR gene is incorporated into the viral DNA


The adenoviruses containing the CFTR gene are then extracted from the epithelial cells, purified and
sprayed into the lungs


Here

they infect the epithelial cells, taking the CFTR gene into the cells


The CFTR channel protein is synthesised as normal


but as the viruses cannot replicate


they do
not damage the cells


Problems include not completely inactivating the genes controlli
ng viral replication and the patients
becoming ill. Also, patients treated repeatedly may develop antibodies than make them immune the
virus and resistant to the treatment


Perfecting Gene Therapy Techniques


Neither of the above methods provides a perman
ent cure for CF but both have the potential to
alleviate some of the symptoms


Before using either method on people, research has to be done to ensure gene therapy is safe


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
9

The first tests, carried out on laboratory animals like mice aimed to find out the
following:


1.

The best way of getting the gene into cells


2.

Check that the gene was expressed in the target cells


3.

Detect any ill effects of the therapy itself


The next step was to start testing the delivery systems on people


This was initially done by gi
ving small doses of liposomes containing the CFTR gene applied to the
lining of the nose to volunteers


(since it is easier to study the cells here than deep in the lungs)


The next stage


which is currently ongoing


involve clinical trials with CF pati
ents


Many trials must be done before gene therapy becomes an officially approved treatment for CF


The Future of Gene Therapy


Progress is proceeding at a very fast rate


Soon it will be possible to


1.

Incorporate a healthy gene into one of the chromosomes
before putting it into a cell


the
chromosomes would be replicated as the cell divides


providing a longer lasting treatment


2.

Treat rapidly dividing cancer cells with genes that kill them


3.

Provide protection for viral infections by inserting genes that i
nterfere with virus replication into
cells


More effective ways of getting genes into cells will make treatment of genetic diseases more effective


The 2 methods described for CF have to be repeated at regular intervals because copies of the gene
are not p
assed form cell to cell during mitosis


Epithelial cells divide frequently so this is a major limitation


If however the healthy gene was inserted into a chromosome then it would be replicated each time
the divided


One problem here though is inserting the

gene into the DNA in a chromosome may damage other
genes


Gene therapy could help in the fight against cancer


There are genes which cause cells death


so if
they could be inserted into cancer cells


tumours cold be destroyed


Reproduction of viruses i
n cells could also be prevented by inserting genes which produce mRNA
that is complementary to the viral RNA


This would inactivate the virus


The most effective treatment for genetic disorders is to replace the defective genes completely

T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
10


This could be do
ne using
in vitro

fertilisation (similar to method for producing transgenic animals)


The dangers of damaging the embryo though means that this technique is not suitable for humans


Such a genetic change would affect the GERMLINE


This means the genes of s
ex cells would be altered and the genetic change would be passed on to
future generations


One other proposed method is to create an artificial chromosome


containing only healthy extra
genes and the promoter genes needed to express them


Once added to t
he nucleus this chromosome would be replicated and take part in mitosis in the
normal way


There is less chance it will cause danger to other chromosomes and genes


Once the full human genome is known further developments will be able to be made


At presen
t there are trials using somatic gene therapy for 100’s of diseases including cancers,
Duchenne muscular dystrophy and haemophilia


Some of the possibilities raise difficult moral and ethical questions


Balancing the benefits for the emerging technology ag
ainst the potential for its abuse will take many
years of consultation and consideration


Genetically Modified Animals


Animals which have been given genes from another species are called TRANSGENIC organisms


Transgenic mammals are useful because they can

express human genes


It is not always possible for bacteria to express human genes since prokaryotic bacterial cells do not
have the ribosomes and other cellular machinery necessary to make complex mammalian proteins


Tracey is the name of a famous transg
enic sheep


When she was an embryo, human genes were transferred into her cells


The human gene to make the human protein alpha


1
-

antitrypsin (AAT) was isolated from a culture
of human cells


The gene was then combined with a promoter sequence that tha
t allows the gene to be expressed in
sheep mammary glands


Mature egg cells were removed from the ovary of a sheep and fertilised
in vitro
. The AAT gene and
its promoter sequence were injected into the nucleus of the fertilised egg cells


Once the zygote
had divided to form a small embryo


this was placed in the uterus of a sheep
surrogate mother


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
11

When the sheep grew to adulthood she was able to produce the protein human alpha


1
-

antitrypsin
(AAT) in her milk. This could then be collected easily and
used


AAT is used to treat emphysema and cystic fibrosis


The normal function of the ATT glycoprotein is to inhibit an enzyme called elastase that is produced
by some types of white blood cell


Elastase, if not inhibited, breaks down the elastic tissue in
the lungs, causing emphysema


In emphysema, the walls of the alveoli disintegrate, so there is less SA available for the absorption of
oxygen and fluid leaks into the air spaces


The fluid disrupts normal functioning of the lungs and can cause infections


Emphysema can be caused by a number of factors.


Having the Z allele for AAT is rare. More common causes are smoking and working with fine dust
particles


There are many different alleles of the AAT gene


About 3% of the population have the Z allele


thi
s codes for a version of AAT that differs from the
normal form by one amino acid. This is a defective form and patients often develop emphysema


Affected people can be treated with an aerosol spray containing AAT


When inhaled this stops the breakdown of
the alveoli and helps breathing


But it is not a permanent cure


patients must have treatment for the whole of their lives


The blood clotting protein Factor VIII is also produced in this way and is used to treat haemophilia


Evaluation of Genetic Enginee
ring


Rapid developments in genetic engineering are making it possible to alter living organisms in a
variety of ways


It is in theory possible to transfer genes from almost any organism to any another


There are many ways in which DNA recombinant technolo
gy can be used in food production and
medicine


However, many people are suspicious of these developments and are concerned about the possible
ways in which the technology might go wrong




There are concerns about the release of genetically engineered organ
isms into the environment.
Once released, these organisms cannot be recalled


No one can be sure of what effect they may
have on other organisms in the environment




Genetically modified crop plants have also caused concern e.g. potatoes have been produce
d
containing a gene which makes them resistant to a herbicide


T.G. AS Module 2


Genes and Genetic Engineering


11.4


Applications of Gene Technology


Teaching Notes
23/03/13

Page
12



This herbicide can be sprayed onto the crop and kills weeds but not the potatoes Might this gene
somehow spread to wild plants and affect the balance of the ecosystem in some way


creating
‘s
uperweeds’.




There are concerns GM foods may be harmful to health




There are concerns genetically modifying crops will INCREASE our current use of pesticides




Will a gene, added to a genome behave in an unforeseen manner? Could it trigger a disease?




Sho
uld the money used to fund research into GE by developed countries be used to provide basic
things like food and clean water in developing countries?




Is DNA fingerprinting reliable? What about contamination of samples used in evidence? (OJ
Simpson trial
)




Are we interfering with nature?




For gene therapy, most people agree that it could be used to relieve the symptoms of a genetic
disease. But the possibility of inserting the ‘correct’ genes into someone’s cells opens up all sorts
of possibilities




In t
heory, any gene could be inserted into the human egg. What types of gene should be
inserted? Designer babies?


At the moment there is a complete ban on performing any genetic engineering on sex cells. This is
likely to continue for the foreseeable futu
re