Download The Living World

Lecture 11
Biotechnology
A Scientific Revolution
 Genetic engineering is the process of moving genes from one
organism to another
 Having a major impact on agriculture & medicine
Restriction Enzymes
 Restriction enzymes bind to
specific short sequences
(usually 4- to 6- bases long) on
the DNA
 The nucleotide sequence on both
DNA strands is identical when GAATTC
read in opposite directions
CTTAAG

Most restriction enzymes cut the
DNA in a staggered fashion
 This generates “sticky” ends
 These ends can pair with
any other DNA fragment
generated by the same
enzyme
 The pairing is aided
by DNA ligase
Play
Restriction Enzymes
4 Stages of a Genetic Engineering Experiment
All gene transfer experiments share four distinct stages
1.
Cleaving DNA
2.
Producing recombinant
DNA
3.
Cloning
4.
Screening
Play
Steps in cloning a gene
Stage 1
 Cleaving the DNA
 The large number of fragments produced are separated by
electrophoresis
Fragments
appear as
bands under
fluorescent light
Stages 2 & 3
 Producing Recombinant DNA
 Fragments of source DNA are inserted into vectors
 Vectors are plasmids or viruses that carry foreign DNA into the host
cell
 Vector DNA is cut with the same enzyme as the source DNA, thus
allowing the joining of the two
 Cloning
 Host cells are usually bacteria
 As each bacterial cell reproduces, it forms a clone of cells
containing the fragment-bearing vector
 Together all clones constitute a clone library
Stage 4
 Screening

A preliminary screen of the
clone library eliminates
1. Clones without vectors
2. Clones with vectors
that do not contain
DNA

The vector employed
usually has genes for
 Antibiotic resistance
 This eliminates the
first type of clones
because they are
sensitive to antibiotics
 b-galactosidase
 This eliminates the
second type of clones
based on X-gal
metabolism and color
changes
Stage 4 (cont.)
 Screening
 To find the gene of interest, the clone library is screened by a process termed
hybridization
 The cloned genes form base pairs with complementary sequences on another
nucleic acid, termed the probe
 The bacterial colonies are first grown on agar
 They are then transferred to a filter
 The filter is treated with a radioactive probe
 The filter is then subjected to autoradiography
Working with DNA
 Key techniques used by today’s genetic engineers include
 PCR amplification
 Used to increase the amounts of DNA
 cDNA formation
 Used to build genes from their mRNA
 DNA fingerprinting
 Used to identify particular individuals
PCR Amplification
Target sequence
Heat
Primers
1 Denaturation

The polymerase chain reaction
(PCR) requires primers


Short single-stranded
sequences complementary to
regions on either side of the
DNA of interest
PCR consists of three basic
steps
Cycle
2
Cool
Cycle
1
2 Annealing of primers
2 copies
3 Primer extension
Heat
4 copies
1. Denaturation
2. Primer annealing
3. Primer extension
Cycle
3
Polymerase Chain Reaction
Cool
Heat
8 copies
Play
DNA polymerase
Free nucleotides
Cool
cDNA Formation
 The primary mRNA
transcript contains
exons and introns
 The processed
mRNA contains only
exons
 It is used as a
template to create a
single strand of
DNA termed
complementary
DNA (cDNA)
 cDNA is then
converted to a
double-stranded
molecule
DNA Fingerprinting
 This is a process that is
used to determine if two
DNA samples are from the
same source
 The DNA from the two
sources is fragmented
using restriction enzymes
 The fragments are
separated using gel
electrophoresis
 They are transferred to a
filter
 The filters are screened
with radioactive probes
 Then subjected to
autoradiography
Play
DNA Fingerprinting
Genetic Engineering and Medicine

Genetic engineering has been used in many medical applications
1. Production of proteins to treat illnesses
2. Creation of vaccines to combat infections
3. Replacement of defective genes
Making “Magic Bullets”
 In diabetes, the body is unable to
control levels of sugar in the blood
because of lack of insulin
 Diabetes can be cured if the body
is supplied with insulin
 The gene encoding insulin has
been introduced into bacteria
 Other genetically engineered drugs
include
 Anticoagulants
 Used to treat heart attack
patients
 Factor VIII
 Used to treat hemophilia
 Human growth hormone (HGH)
 Used to treat dwarfism
Piggyback Vaccines
 Genetic engineering has also been used to create subunit vaccines
against viruses
A gene encoding a viral
protein is put into the DNA of
a harmless virus and
injected into the body
The viral protein will elicit
antibody production in the
animal
A novel kind of vaccine was
introduced in 1995
The DNA vaccine uses
plasmid vectors
It elicits a cellular immune
response, rather than
antibody production
Genetic Engineering of Farm Animals

In 1994, the recombinant hormone bovine
somatotropin (BST) became commercially
available
 Dairy farmers used BST as a supplement to
enhance milk production in cows

Consumers are concerned about the presence of
the hormone in milk served to children
 This fear has not been supported by research data
Genetic Engineering of Crop Plants

Pest resistance
 Leads to a reduction in the
use of pesticides
 Bt, a protein produced by
soil bacteria, is harmful to
pests but not to humans
 The Bt gene has been
introduced into tomato
plants, among others

Herbicide resistance
 Crop plants have been
created that are resistant to
glyphosate

Herbicide resistance offers two main advantages
 Leads to a reduction in the use of pesticides
 Lowers the cost of producing crops
 Reduces plowing and conserves the top soil
Genetic Engineering of Crop Plants
 More Nutritious Crops
 Worldwide, two major deficiencies are iron and vitamin A
 Deficiencies are especially severe in developing countries where the
major staple food is rice
 Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden” rice
to solve this problem
Potential Risks of Genetically Modified (GM) Crops

The promise of genetic engineering is very much in evidence


However, it has generated considerable controversy and protest
 Are genetic engineers “playing God” by tampering with the genetic
material?
Two sets of risks need to be considered
1. Are GM foods safe to eat?

The herbicide glyphosate blocks the synthesis of aromatic amino acids
 Humans don’t make any aromatic amino acids, so glyphosate doesn’t
hurt us

However, gene modifications that render plants resistant to glyphosate
may introduce novel proteins
 Moreover, introduced proteins may cause allergies in humans
2. Are GM foods safe for the environment?

Three legitimate concerns are raised
1. Will other organisms be harmed unintentionally?
2. Will pests become resistant to pesticides?
3. What if introduced genes will pass from GM crops to their wild or
weedy relatives?
Potential Risks of Genetically Modified (GM) Crops
 Should GM foods be labeled?
Every serious scientific investigation
has concluded that GM foods are
safe
So there is no health need for a GM
label
However, people have a right to know
what is in their food
So there may be a need for label
after all
Cloning Higher Organisms
 The successful embryos (about 30 in 277 tries) were transplanted into
surrogate mother sheep
 On July 5, 1996, “Dolly” was born
 Only 1 of 277 tries succeeded
 However, Wilmut proved that reproductive cloning is possible

Since Dolly,
scientists have
successfully cloned
sheep, mice, cattle,
goats and pigs
 However, problems
and complications
arise, leading to
premature death
 Dolly died in 2002,
having lived only
half a normal
sheep life span
Embryonic Stem Cells



The blastocyst, an early
embryo, consists of
 A protective outer layer that
will form the placenta
 Inner cell mass that will
form the embryo
The inner cell mass consists of
embryonic stem cells
 These are pluripotent
 Capable of forming the
entire organism
As development proceeds,
cells lose their pluripotency
 They become committed to
one type of tissue
 They are then called adult
stem cells

The research in human embryonic stem cells is
associated with two serious problems
 Finding a source: harvesting them from
discarded embryos raises ethical issues
 Immunological rejection: Implanted stem
cells will likely be rejected by the immune
system of the individual
Stem Cells
 Embryonic stem cells
could be used to restore
tissues lost or damaged
due to accident or
disease
 Experiments have
already been tried
successfully in mice
 Damaged spinal
neurons have been
partially repaired
 The course of
development is broadly
similar in all mammals
 Therefore, the
experiments in mice
are very promising
Grappling with the Ethics of Stem Cell Research

Stem cells offer enormous promise for treating a wide range
of diseases
 However, the research involves ethical issues
1. Destruction of human embryos
 When does human life begin?
2. Possibility of future abuse or misuse
 Is human reproductive cloning next?
3. Alternative sources of stem cells
 Are adult stem cells equally effective?
Gene Therapy
 Gene therapy involves the
introduction of “healthy”
genes into cells that lack
them
 It was first used successfully
in 1990
 Two girls were cured of a
rare blood disorder caused
by a defective adenosine
deaminase gene
 The girls stayed healthy
 In 1999, AAV successfully cured anemia in rhesus monkeys
 AAV was also used to cure dogs of a hereditary disorder leading to retinal
degeneration & blindness