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DNA TECHNOLOGY
Developments and Applications
Genetic Engineering
• Modifying an organism’s genotype by
introducing genes that have never been
present in the chromosomes of that
particular species.
HISTORY
• 1952- Birth of a calf by artificial insemination (AI)
• 1953- Watson & Crick’s model of DNA
• late 1960s- Restriction enzymes in E. coli were
discovered to 'cut' DNA at specific sites.
• 1969- Harvard Medical team first to separate a gene
from DNA
• 1972- First animal born from frozen embryo
• 1973- First use of restriction enzymes to insert DNA
into a plasmid and make many copies of the DNA.
• 1977-Walter Gilbert and Frederick Sanger worked
out methods to determine the sequence of bases in
DNA
History (cont.)
• 1978- Birth of Louise Brown-- the first 'test-tube'
baby born from in vitro fertilization (IVF)
• 1979- First production of insulin through genetic
engineering.
• 1980- First transgenic (genetically modified) mouse.
• 1982- Giant mouse produced by transferring growth
hormone genes from a rat
• 1983- First transgenic plant (tobacco)
• 1984-Birth of a baby from an embryo which had been
stored frozen and development of DNA fingerprint.
• 1988- First transgenic plant producing a
pharmaceutical & first animal patented (oncomouse)
by Harvard Univ.
Transgenic OrganismAn organism
whose genome
has been
modified by
introduction of
novel DNA
Transgenic Plants
“Glowing” Transgenic Plants
This "autoluminograph" of a glowing transgenic tobacco plant
bearing the luciferase gene of a firefly was first done in 1986
More History
• 1990- Human Genome Project started
• 1991- first gene therapy trials on humans
• 1992- US military begins collects blood samples of all
soldier to serve as genetic “dog tags”
• 1994- first transgenic tomatoes sold in stores (with
softening inhibition gene)
• 1997- Dolly was born (first animal cloned by nuclear
transfer using a mammary cell from an adult sheep as
nucleus donor and an enucleate ovum as recipient).
 1998 - Researchers first extract stem cells from human
embryos
• 1999- First publicly reported human death from gene
therapy.
2000’s
• 2000- Human genome draft completed
• 2001
– Birth of first genetically modified primate, a rhesus monkey
named ANDi (inserted DNA backwards) and containing a fluorescent
marker gene from a jellyfish, at Oregon Regional Primate Research
Centre, USA
– Publication in the journals Nature and Science of the first draft of the
human genome estimated to be between 26,000 and 40,000 genes.
– President Bush restricts federal funding for embryonic stem-cell
research
• 2002- First cloned cat (cc)
• 2003- Dolly is euthanized after developing progressive lung disease
• 2004– South Koreans claim first cloned human
– Harvard researchers grow stem cells from embryos using private
funding
– Ballot measure for $3 Billion bond for stem cells research
• 2005- South Koreans exposed for lying about human clone
• 2009- Obama overturns stem cell ban, but opposes human cloning
A little background on cloning
and stem cell technology
Different Types of Stem Cells
You recall sexual
reproduction
The Association of Reproductive Health Professionals
Embryonic Stem Cells
Derived from embryos
that develop from eggs
that have been
fertilized in vitro and
then donated for
research purposes
with informed consent
of the donors.
They are not derived
from eggs fertilized in
a woman's body
Princeton University
Blastocyst Diagram
Princeton University
University of Wisconsin-Madison
Adult Stem Cell
•An undifferentiated cell
found in a differentiated
tissue that can renew itself
and differentiate (with certain
limitations) to give rise to all
the specialized cell types of
the tissue from which it
originated.
•It is important to note that
scientists do not agree about
whether or not adult stem
cells may give rise to cell
types other than those of the
tissue from which they
originate.
http://stemcells.nih.gov/info/basics/basics1.asp
What’s Cloning?
• Cloning is the creation of an organism that
is an exact genetic copy of another.
Human Clones?
In nature, twins
occur just after
fertilization of an
egg cell by a
sperm cell.
Each cell continues
dividing on its own,
ultimately
developing into a
separate individual
within the mother.
In rare cases, a
zygote tries to
divide into a twocelled embryo, the
two cells separate.
Since the two cells
came from the same
zygote, the resulting
individuals are
genetically identical.
Are there human clones?
Yes – Identical Twins
(Time 2.19.01)
But Not Really……
A tale of
two sisters
• According to new research, though identical twins share
very similar genes, they actually ARE NOT identical.
– It has long been known that identical twins develop
differences that result from the environment
– Now scientist recognize Epigenetic factors:
• The chemical markers that attach to genes and affect how
they are expressed– modifying genes
• Basically “genetic changes that occur after the parents’
template had been laid”.
• They accumulate over a lifetime and arise from things like
diet and tobacco use.
• They have been implicated in the development of cancer and
behavioral traits like fearfulness and confidence.
• They can slow or shut genes off or increase their output
• These markers vary widely from one person to another
Calm rats
• How does a high-nurturing mother rat shapes her pup's
epigenome, and what that pup's response to stress will
be.
• In rats, does licking by the mother activate, or deactivate
her pup's GR gene?
• Explain how cortisol and the GR protein work together in
the brain to relax a rat pup.
• The rat nurturing example shows us how parental
behavior can shape the behavior of their offspring on a
biochemical level. Relate this to humans and think about
the personal and social implications.
Artificial embryo twinning
• Uses the same approach as natural identical twins, but it
occurs in a Petri dish instead of in the mother's body.
• This is accomplished by manually separating a very early
embryo into individual cells, and then allowing each cell
to divide and develop on its own.
http://www.abpischools.org.uk/resources/posterseries/images/clon02.png
Asexual Reproduction
The Association of Reproductive Health Professionals
Somatic Cell Nuclear Transfer
• Uses a different approach than artificial embryo
twinning, but it produces the same result: an
exact clone, or genetic copy, of an individual.
Involves
removing the
maternal
nucleus before
nuclear
transfer
(Roslin Institute http://www.roslin.ac.uk/library/)
Nuclear transfer
Nuclear transfer embryo about to be
activated
(Roslin Institute http://www.roslin.ac.uk/library/)
Cloning A Sheep
•Ian Wilmut and his
colleagues
replaced the
nucleus of a
sheep’s egg with
the nucleus of a cell
from a sheep’s
udder.
• They grew the
udder cells in a lab
and to remove the
genetic blockages
created during
differentiation, they
used the nucleus of
a cell in the G0
phase of the cell
cycle.
(Roslin Institute http://www.roslin.ac.uk/library/)
•Farmers and
scientist see the
agricultural value of
cloning animals b/c
one prized animal
could be replicated
many times without
the random
changes that occur
with two parents.
•However, nuclear
transfer produces a
live offspring in less
than one in every
100 attempts.
Click & Clone
• http://learn.genetics.utah.edu/content/tech/
cloning/
Cloning techniques for
creating stem cells
Therapeutic cloningDuplicating part of a person e.g. a
heart or liver, or even just a few cells.
Reproductive cloningDuplicating a person e.g. identical
twins.
http://biochem118.stanford.edu/images/Stem%20Cell%20Slides/10.%20Therapeutic%20Cloning.jpg
•Taking genetic material from the patient and putting it into a donated egg.
•This has tremendous therapy potential because the human immune system
is less likely to attack genetically identical cells.
•The resultant cells would be a perfect match for the individual and could
mean treatments for diseases like diabetes without problems of rejection.
•Experts warned that there was a risk the cells could become cancerous.
Is a clone genetically identical?
• Almost – but not quite.
• Even identical twins are different due to the slight
differences in environment they experience.
• Clones would be even more different as they
would live in a very different environment and time
as a form of “delayed identical twin”.
Potential uses of cloning
•
•
•
•
•
•
•
Replacing organs and other tissues
Infertility
Replacing a lost child
Creating donor people
Gene therapy
Saving endangered species
Reversing the ageing process
Problems with cloning?
•
•
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•
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•
•
•
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Low success rate (Dolly took 276 attempts)
Tumors
Genetic defects
Over-growth syndrome
Pre-mature ageing (genetic age)
Massive quantities of human eggs required
Reduction in adaptability – genetic uniformity
Insertion of genes
Lack of knowledge
IVF – In Vitro Fertilization




Available since 1986
About 100,000 IVF kids in U.S.
Often creates extra embryos
U.S. - About 400,000 frozen embryos
(unregulated)
Recent News
• On August 23, 2006, Nature, the scientific journal, published a letter
by Dr. Robert Lanza, claimed that his team had found a way to extract
embryonic stem cells without destroying the actual embryo.1
– This would enable scientists to work with new lines of embryonic stem
cells derived using public funding.
• On 20th of November, 2007, two research teams announced a similar
breakthrough with ordinary human skin cells that were transformed
into batches of cells that look and act like embryonic stem cells. 2
• On Dec. 6th, 2007 a research team at the Whitehead Institute claimed
to cure mice of sickle cell anemia. 3
• However, the genes used to reprogram the skin cells were added by
the use of retroviruses that can cause mutations and possibly lead to
the risk cancers.4
• On January 16, 2008, a California based company, Stemagen,
announced that they had created the first mature cloned human
embryos from single skin cells taken from adults.5
1.
Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R. (2006). "Human embryonic stem cell lines derived from single blastomeres.". Nature
444 (7118): 481-5. PMID 16929302. 2 "Embryonic stem cells made without embryos", Reuters, November 21, 2007.
3 Rick Weiss. "Scientists Cure Mice Of Sickle Cell Using Stem Cell Technique: New Approach Is From Skin, Not Embryos", Washington Post,
December 7, 2007, pp. A02.
4 "Researchers get closer to safe stem cell treatments", AFP, February 14, 2008.
5 Helen Briggs. "US team makes embryo clone of men", BBC, January 17, 2008, pp. A01.
More Recent News
• January 23, 2009-- National Geographic reported that the
journal Theriogenology describes the use of frozen skin in
2003 to clone a bucardo, or Pyrenean ibex, a subspecies of
Spanish ibex that went extinct in 2000.
– But David Wildt, a senior scientist at the Smithsonian's National Zoological
Park in Washington, D.C., who did not participate in this study, warned that the
successful cloning wouldn't be the first step in creating a real-life Jurassic
Park.
– "The public should not leap to the conclusion that we are on the edge of
cloning woolly mammoths or dinosaurs," he said. "Even if such embryos could
be constructed, there are no appropriate surrogate mothers for long-dead
species."
• March 9, 2009-- Obama Says Government Will Not Open the
Door for Human Cloning
– President Obama says the government will not open the door for
human cloning, as he signed an order lifting restrictions on federal
funding for embryonic stem cell research.
RECOMBINANT DNA
• molecules that contain DNA sequences derived
from different biological sources that have been
joined together in the laboratory.
Genetically engineered food:
•Insecticide sweet corn- Scientists have genetically modified
sweet corn so that it produces a poison which kills harmful
insects.
•Golden rice – large amounts of vitamin A from gene in carrots
adds nutrients to rice.
•Frostban strawberries- protects strawberries from natural
bacteria that cause frost to form
•Pesitcide resistant plants- When the farmer sprays his
genetically modified crop with pesticides, he or she can destroy
most of the pests without killing the plants
Genetically engineered
animals:
• Mice with human proteins- like
oncomouse (has a gene for cancer)
• Pharming- livestock have been
produced that express foreign proteins
in their milk (like Factor IX or insulin).
– A pig expressing human protein C (a clot-busting drug) in
its milk is worth about $1,000,000 per year.*
• AquaAdvantage© Salmon – grow from
egg to market size in half the time
*”Pig-napping may become a more common crime, as the pharming
develops”
from http://www.escience.ws/b572/L21/L21.htm
VOCAB to KNOW for this Unit
TRANGENIC- an organism that carries a foreign gene that has been
deliberately inserted into its genome.
SPLICING– inserting the gene in question into a larger molecule of DNA
•VECTORtransport vehicle which
carries the desired gene
into the host cell.
•PLASMIDa small circular DNA molecule
found in bacteria (separate from
the main bacterial
chromosome).
More Vocab
• RESTRICTION ENZYME- - a
special DNA-cutting enzyme
that is found in bacteria
• DNase- eukaryotic DNAdigesting enzymes
• DNA LIGASE- an enzyme that
seals the gaps in the DNA
• DNA CLONING- making many
copies of DNA (amplification)
through the use of a host
bacterial cell
And still some more Vocab……
• RESTRICTION FRAGMENTS- Pieces of DNA of varying
lengths
•GEL ELECTROPHORESIS- DNA fragments move through
a porous gel in response to an electric current (small = fast)
•DNA PROBE- a single-stranded DNA fragment that
possesses a particular sequence which is complementary
to the fragment being sought. Sort of like a guided missile.
POLYMERASE CHAIN REACTION (PCR)- a way of
amplifying a single region of DNA using DNA polymerase
and heat.