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Transgenic cow research
at AgResearch
Liz Carpenter
Dairy Biotechnology
Contents of the Talk
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Introduction to AgResearch
Terminology
Steps to develop a transgenic
Biological implications
Ethical implications
Questions
Introduction to AgResearch
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One of the largest life sciences companies in
the Southern hemisphere
Employs > 1300 staff
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Have science capabilities in:
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Plant genes
Animal genes
Food and health
Agri-technologies
Sustainable resource management
Transgenic programme
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Aim:
produce high value proteins, for
use in human pharmaceuticals,
in cow’s milk
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First transgenic calves born in 2000
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Approx. 25 people involved in the
generation of successful transgenics
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Planning and research has
taken more then 6 yr
Terminology (i)
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Genome of an organism
The set of chromosomes, containing all
the genes and associated DNA.
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DNA (deoxyribonucleic acid)
String of nucleotides (bases A, T, G, C)
which carries the genetic information of
a cell. Carries the necessary
information to construct proteins.
Terminology (ii)
 Gene
Segment of DNA that codes for a
protein
 Cloning
Creates a whole organism from a
single cell
This organism will have all the same genes as the
original cell…including any genetic modifications
Terminology (iii)
 Transgenic animal
DNA from one species, or
altered DNA from the same species,
is introduced into the genome
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
Design transgene
Transform bovine cells with transgene
Select for transgene positive cells
Clone with genome from these cells
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Increased casein production in milk leads to:
- increased calcium in milk
- improved processing for cheese
Human-derived myelin basic protein (MBP)
produced in milk could be used as a
therapeutic for people with Multiple Sclerosis
(MS)
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
- A bovine genome library was screened by PCR
for colonies containing the casein DNA sequence
- Plasmid DNA was extracted as a source
of the casein DNA sequence
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
The DNA sequence is cut out of the
bacterial plasmid with specific
restriction enzymes
There are over 100 restriction enzymes,
and each cuts DNA at a specific nucleotide
sequence
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
Design transgene
The gene must be adapted to be
expressed in the bovine mammary gland
How to make a GMO cow
in 7 basic steps
The transgene is made up of the desired
gene, bracketed by a:
a)selectable marker gene
b)promotor sequence
c) termination sequence
Selection
Start
Desired gene
Stop
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
Design transgene
Transform bovine cells with transgene
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
Design transgene
Transform bovine cells with transgene
Select for transgene positive cells
- Cells are incubated with an antibiotic
- Cells which have the transgene integrated into
the chromosome will survive
Selection
Cell carrying
transgene
+ antibiotic for 2 days
Check for presence of transgene by PCR & Southern blot
PCR
(polymerase chain reaction)
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DNA unzips when heated
Single strands of complementary DNA
bind together (A – T, G – C, T – A, C – G)
DNA polymerase repairs second strand of
DNA (assuming free nucleotides present)
http://www.biotech.iastate.edu/
Principles of PCR
http://allserv.rug.ac.be/~avierstr/principles/pcr.html
Principles of PCR
http://allserv.rug.ac.be/~avierstr/principles/pcr.html
It’s my 17th birthday on Saturday and I’m
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Can you ring two people for me ?
Cycle:
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http://www.biotech.iastate.edu/
Principles of PCR
http://allserv.rug.ac.be/~avierstr/principles/pcr.html
Southern Blot
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Transformed cell DNA is extracted
DNA fragments are separated by gel
electrophoresis
DNA is transferred onto a nitrocellulose filter
Specific DNA fragments are detected by
hybridization to radioactive probes
(Non-radioactive techniques are also available)
1 2 3 4 5
http://www.accessexcellence.org/AB/GG/southBlotg.html
How to make a GMO cow
in 7 basic steps
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Identify trait (gene) of interest
Extract DNA (with gene) from source
Isolate gene sequence
Design transgene
Transform bovine cells with transgene
Select for transgene positive cells
Clone with genome from these cells
Transgenic Cows 2003
How to show a GMO cow
is expressing the gene of interest in milk
WT
A1 A2 A3
b-casein
Casein plus
A1 A2 A3
b-casein
Genetic background
b-casein A1A2
Transgene
b-casein A3
Biological Implications
Benefits of GMO animals
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Casein cows - milk requires less processing
» - less environmental pollution
- increased natural calcium levels
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BioPharming: Cows make 20 litres milk/day
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Enviropigs:
Digest phosphorous efficiently
Reduce P in manure and runoff
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Disease resistance eg mastitis (bacteria in milk)
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Xenotransplantation (using animal tissues for human
transplantation)
Biological Implications
Risks of GMO animals
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Horizontal Gene Transfer ?
– What is the risk ?
– Can it happen?
» Whole transgene & promotor must be released
» DNA is easily broken down (6 hr in soil)
» Transgene must be taken up whole
» Transgene must be integrated into new organism
» Mammalian DNA can’t be “read” by bacteria
» Transgene must give new host some benefit for it
to be maintained – is this likely ?
Biological Implications
Risks of GMO animals
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Mixing with non-GMO animals
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May get into the human food chain
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AgR maintains secure Containment Facilities
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No transgenic animals leave the facility EVER !
Containment Facility – main gate
Access
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No Unauthorised Entry
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Access procedures displayed in entrance
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No unsupervised access without training
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Visitors allowed with Operator’s and/or
Managers consent
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Daybook
Perimeter double fence
Ethical Implications
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Science deals with testable facts
Ethics considers right & wrong:
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Not testable
May change between cultures / religions
May change over time (with new discoveries)
Do we need to be scientific experts to have an
ethical opinion about GE ? (e.g. consider nuclear
weapons)
Ethical Implications
Two main types of ethical issues for GMOs
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Extrinsic issues, ie consequences of application
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Potential benefits & harms / risks
(Uncertainty – facts vs beliefs)
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Human & animal welfare
Environment – are we changing the world ?
Intrinsic issues - issues of rights,
responsibilities
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Include religious, spiritual, cultural beliefs
The morality continuum:
where do you stand?
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All genetic engineering is bad
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Insulin produced by GM bacteria saves lives
My mother is a diabetic. She would die if she relied on
pig insulin because it makes her sick.
I can't afford to buy insulin so now I am blind.
Where does it come from?
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Labelling is too difficult for bulk products
The cost of keeping GM products separate outweighs
any commercial gain
I will have an allergic reaction to food with any traces of
peanut. It is a matter of life or death whether food is
labelled correctly.
The morality continuum:
where do you stand?
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What is the science involved?
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There is no DNA in the oil from GM conola plants.
Therefore the product is GM free.
Cows which eat GM plants break down the DNA in the
gut. Therefore the cows are GM free.
The foreign gene is only expressed in the leaves of
canola. This means the seeds are GE free.
Questions