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Cloning
Gene cloning – replication of donor genes in
bacterial or other host cells
– clone – a group of cells, organisms, or genes that are
exact copies of each other
– donor gene inserted into a bacterium is copied every
time the plasmid containing it replicates – genes can be
cloned by growing genetically engineered bacteria
Controlling gene expression – genetic engineers
want to be sure that only bacteria with donor gene
replicate
– to ensure this, a gene for antibiotic resistance is
attached to donor gene and antibiotic is used to kill all
unwanted bacteria that do not have the donor gene
Polymerase Chain Reaction
• cloning a gene through genetic engineering can be timeconsuming and requires an adequate DNA sample as
starting material
• PCR technique allows researchers to amplify a tiny sample
of DNA millions of times in a few hours
• DNA polymerase uses nucleotides and primers to replicate
a DNA sequence in vitro, thereby producing two molecules
• Two strands of each molecule are then separated by
heating and replicated again, so then there are four,
double-stranded molecules
• After the next cycle of heating and replication there are
eight molecules, and so on
• Number of molecules doubles with each cycle
• PCR is useful in amplifying tiny samples of DNA ranging
from crime scenes to archaeological remains
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Cloning organisms – cloning sometimes
occurs naturally (twins, asexual
reproduction)
organisms can be cloned artificially
(sheep, rabbits, toads and other sexually
reproducing animals have been cloned by
dividing up an embryo and transplanting
them into surrogate mothers)
Cloning of Dolly
1. sheep cloned from a non-reproductive cell
2. cell taken from udder of donor adult and cultured
in lab for 6 days
3. unfertilized egg taken from another sheep –
nucleus removed
4. egg without nucleus is fused with donor cell using
a spark of electricity
5. embryo resulting from fusion of udder cell and egg
transferred into the uterus of a third sheep who
acts as the surrogate mother
6. surrogate mother gives birth to lamb – lamb is
genetically identical to sheep that donated udder
cell
Human Genome Project
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A project that involved mapping the entire human
genome – determined the order of all the bases in
human DNA
Outcomes of the HGP:
1. Determine how many individual genes we have and
how they work.
2. Locating and determining the cause of genetic
disorders.
3. Development of gene therapies to treat genetic
disorders.
4. Comparing genetic makeup of human populations to
determine ancestries and how humans have migrated
and mixed their genes with other populations over
time.
Practical Applications of DNA
Technology
•
DNA technology is reshaping medicine
and the pharmaceutical industry
–
one obvious benefit of DNA technology and
of the Human Genome Project (international
cooperative venture established to sequence
the complete human genome) is the
identification of genes whose mutation is
responsible for genetic diseases
Diagnosis of Disease and Genetic Screening
(testing an individual for the presence or
absence of a gene)
– medical scientists currently use DNA
technology to diagnose hundreds of human
genetic disorders and to detect other diseases
such as HIV in blood or tissue samples
(important to keep blood supply safe)
– this allows early disease detection and
identification of carriers for potentially harmful
recessive mutations – even before the onset of
symptoms
• genes have been cloned for many genetic
disorders including hemophilia,
phenylketonuria, cystic fibrosis, and
Duchenne muscular dystrophy – this allows
direct detection of gene mutations
– cloned normal gene can be used as a probe to
find the corresponding gene in cells being
tested
• alleles for cystic fibrosis and Huntington’s
disease can also be detected
there are advantages and disadvantages to
genetic screening
• advantages include pre-natal diagnosis of
genetic diseases (before the baby is born),
ability to detect carriers of genetic diseases,
confirmation of animal pedigrees (DNA
testing required in dogs), resolution of
immigration disputes
• disadvantages include ethical issues – i.e.
question of abortion if fetus is found to have
a genetic disorder
Human Gene Therapy – example: Cystic Fibrosis
• theoretically, it should be possible to replace or
supplement defective genes with functional normal
genes using recombinant DNA techniques
• CF is the most common inherited disease among
northern Europeans and white North Americans
• due to a recessive gene located on chromosome 7
• normal allele controls the production of a
membrane protein called cystic fibrosis
transmembrane protein (CFTP) – essential for
proper functioning of epithelial cells (normal
epithelial mucus is watery) which traps dust and
microorganisms – mucus moves particles up throat
to be swallowed or blown out of nose
• CF patients have abnormally thick mucus –
difficult to clear, clogs airways, breeding
ground for bacteria
• lack of CFTP also causes growth of fibrous
cysts in pancreas which interfere with
delivery of digestive enzymes
• CF gene has been cloned allowing for
identification of causes of disease, allowing
for genetic screening, and possible gene
therapy
• gene therapy for CF targets the lungs – gene for
normal CFTP is isolated and inserted into a
bacterial plasmid
• plasmid is introduced in bacterial cell for cloning by
bacteria (i.e. multiple copies are made)
• plasmids are inserted into liposomes (sphere of
lipid that can penetrate the cell surface membrane)
• liposomes are taken as nasal spray
• liposomes enter cells of lung tissue; normal allele
is expressed
• treatment only effective for a few weeks as
epithelial cells lining the respiratory tract are shed
regularly
Pharmaceutical Products
– DNA technology has been used to make
human insulin and growth hormone
Forensic uses – forensic labs can determine
blood or tissue type from blood, small
fragments of other tissue, or semen left at
the scene of violent crimes
– DNA fingerprinting can be used for
identification of criminals
Environmental uses
• genetically engineered microorganisms that
can extract heavy metals from their
environment
• genetically engineered microbes may be
used in mining and in cleaning up mining
waste
• bacterial strains have been developed to
detoxify specific wastes found in spills and
waste dumps
Agricultural uses of DNA technology
Animal Husbandry – many farm animals are
treated with products made by recombinant
DNA methods (examples include vaccines,
antibodies, and growth hormones)
– some milk cows are injected with bovine
growth hormone (BGH) made by E. coli, in
order to raise milk production
– BGH also improves weight gain in beef
cattle
Transgenic animals – animals that contain genes from
another species have been developed for agricultural use
(examples include beef and dairy cattle, hogs, sheep and
several species of commercially raised fishes)
– modified DNA can be introduced into diary cows so that
they produce human proteins – protein is produced in
the milk – examples of medically important proteins that
have been produced in transgenic mammals include:
• blood clotting Factor VIII to treat hemophilia
• alpha-1- antitrypsin which helps protect the lungs
from damage during infections
– rainbow trout and salmon that are given a foreign
growth hormone can reach in one year a size that
usually requires 2 to 3 years of growth
Genetic engineering in plants
– plants have been genetically altered to
receive herbicide resistance (several
strains of cotton) – allows them to be
resistant to herbicides used to kill weeds
– some crop plants are being engineered to
resist infectious pathogens and pest
insects – reduces need to apply chemical
insecticides
• first genetically engineered fruits approved
by the FDA for human consumption were
tomatoes engineered with antisense genes
that retard spoilage
– researchers isolated gene responsible for
ripening
– they prepared a gene who's template strand had
a base sequence complementary to the normal
gene – an antisense version of the gene
– when spliced into the DNA of a tomato plant, the
antisense gene is transcribed into RNA that is
complementary to the ripening gene’s mRNA
– the antisense RNA binds to the normal mRNA,
blocking the synthesis of the enzyme causing
ripening and spoilage
Benefits and Possible Harmful
Effects of Genetic Modification
• See handouts and Clegg pg. 127-128
Stem Cells
• Cells that retain their ability to divide and
differentiate into various cell types
• Plants contain stem cells in their meristems
(reason why a cutting can grow into a new
plant)
• Embryonic stem cells are pluripotent – can
form any type of cell in an organism or can
form a complete organism
• Adult stem cells can divide to form new
body tissue cells (i.e. blood stem cells)
Therapeutic Uses of Stem Cells
• Embryonic stem cells are the most flexible
and can grow into any type of mature cell
– Parkinson’s and Alzheimer’s Disease can be
potentially treated by implanting stem cells that
could replace the damaged cells
Ethical Issues surrounding
therapeutic cloning
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Therapeutic Cloning is the creation of an embryo to
supply embryonic stem cells for medical use
– Raises issue of whether it is right or wrong to
generate a new human embryo for medical
research
Two distinct forms of cloning:
1. Reproductive cloning – making copies of entire
organism
2. Therapeutic cloning – making copies of embryonic
stem cells
Opinions vary about whether both forms are
right/wrong or if one or the other is acceptable