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 Organism-

An 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

Princeton University

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

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?

Are there human clones?

Yes – Identical Twins(Time 2.19.01)

In nature, twins occur just after fertilization of an egg cell by a sperm cell.

In rare cases, a zygote tries to divide into a two-celled embryo, the two cells separate.

Each cell continues dividing on its own, ultimately developing into a separate individual within the mother.

Since the two cells came from the same zygote, the resulting individuals are genetically identical.

But Not Really……• 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

A tale of two sisters

• 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.

Calm rats

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/poster-series/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

(Roslin Institute http://www.roslin.ac.uk/library/)

•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.

•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 cloning-Duplicating part of a person e.g. a heart or liver, or even just a few cells.

Reproductive cloning- Duplicating 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?

• 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

SPLICING- – inserting the gene in question into a larger molecule of DNA

•VECTOR- transport vehicle which carries the desired gene into the host cell.

•PLASMID- a small circular DNA molecule found in bacteria (separate from the main bacterial chromosome).

TRANGENIC-

- an organism that carries a foreign gene that has been deliberately inserted into its genome.

More Vocab• RESTRICTION ENZYME- - a

special DNA-cutting enzyme that is found in bacteria

• DNase- eukaryotic DNA-digesting 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.