Transgenic cow research at AgResearch

Liz CarpenterDairy Biotechnology

Contents of the Talk

Introduction to AgResearch Terminology Steps to develop a transgenic Biological implications Ethical implications Questions

Introduction to AgResearch

One of the largest life sciences companies in the Southern hemisphere

Employs > 1300 staff

Have science capabilities in: Plant genes Animal genes Food and health Agri-technologies Sustainable resource management

Transgenic programme

Aim: produce high value proteins, for use in human pharmaceuticals, in cow’s milk

First transgenic calves born in 2000

Approx. 25 people involved in the generation of successful transgenics

Planning and research has taken more then 6 yr

Terminology (i)

Genome of an organism

The set of chromosomes, containing all the genes and associated DNA.

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 cowin 7 basic steps

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 cowin 7 basic steps

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 cowin 7 basic steps

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 cowin 7 basic steps

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 cowin 7 basic steps

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 cowin 7 basic steps

The transgene is made up of the desired gene, bracketed by a:

a)selectable marker gene, e.g. antibiotic

b)promotor sequence

c) termination sequence

Start StopSelection Desired gene

How to make a GMO cowin 7 basic steps

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 cowin 7 basic steps

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)

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 PCRhttp://allserv.rug.ac.be/~avierstr/principles/pcr.html

Principles of PCRhttp://allserv.rug.ac.be/~avierstr/principles/pcr.html

It’s Mum’s 40th birthday on Saturday and we’re having a surprise party at 8 pm.

Can you ring two people for me ?

Cycle: 3 4 5 6

8 16 32 64

128 250 500 1000 2000 4000 8000

16000 32000 64000 128000 256000 500000 106

7 8 9 10 11 12 13

14 15 16 17 18 19 20

Principles of PCRhttp://allserv.rug.ac.be/~avierstr/principles/pcr.html

http://www.biotech.iastate.edu/

Southern Blot

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 cowin 7 basic steps

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 cowis expressing the gene of interest in milk

b-casein

A1A2 A3

Casein plus

Transgene b-casein A3

WT

b-casein A1A2Genetic background

A1 A2

b-casein

A3

Biological Implications

Benefits of GMO animals Casein cows - milk requires less processing

» - less environmental pollution

- increased natural calcium levels

BioPharming: Cows make 20 litres milk/day

Enviropigs: Digest phosphorous efficiently Reduce P in manure and runoff

Disease resistance eg mastitis (bacteria in milk)

Xenotransplantation (using animal tissues for human transplantation)

Biological Implications

Risks of GMO animals 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

Mixing with non-GMO animals

May get into the human food chain

AgR maintains secure Containment Facilities

No transgenic animals leave the facility EVER !

Containment Facility – main gate

Access

No Unauthorised Entry Access procedures displayed in entrance No unsupervised access without training Visitors allowed with Operator’s and/or

Managers consent Daybook

Perimeter double fence

Ethical Implications

Science deals with testable facts Ethics considers right & wrong:

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 ImplicationsTwo main types of ethical issues for GMOs

Extrinsic issues, ie consequences of application Potential benefits & harms / risks

(Uncertainty – facts vs beliefs) Human & animal welfare Environment – are we changing the world ?

Intrinsic issues - issues of rights, responsibilities Include religious, spiritual, cultural beliefs

The morality continuum: where do you stand?

All genetic engineering is bad 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? 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?

What is the science involved?

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