1. Jessica Götzfried Methods Seminar 03.12.14
Transgenic Animals
Jessica Götzfried
Methods Seminar

2. Overview Introduction Historical Background
Transgenesis – The method for the production of transgenic animals
Examples for the use of transgenic animals
Ethical concerns

3. Introduction
A transgenic animal carries a foreign gene that has been deliberately inserted into its genome.
Official term: GMO’s (Genetically Modified Organism)
Transgene – exogenous DNA that is introduced into the animal
Transgenesis is the overall process of introducing exogenous DNA stably into the genome of an animal.
These foreign genes are inserted into the germ line of the animal, so they can be transmitted to the progeny.

4. Why do we need transgenic animals?
Research
Studying gene expression/gene function
Establishing models for studying human diseases (Knock in / knock out mice)
As bioreactors:
Within the field known as pharming, intensive research has been conducted to develop transgenic animals that produce biotherapeutics
(vaccines, antibodies, proteins…)
Agricultural importance:
Disease resistant animals
Improvement of
Milk yield
Wool characteristics
Rate of weight gain
Egg-laying frequency

5. Historical background
1966 – First report of microinjection of mouse embryo
1981 – First transgenic mouse (Gordon and Ruddle) – first time the term transgenic occured
The ‘Supermouse’ produced by Brinster and Palmiter
It was created by inserting a human growth hormone gene in mouse genome.
The offspring was much larger than the parents.
1983 – Tissue specific gene expression in transgenic mice
1987 – Transgenic pig, sheep, rabbit and fish
1988 – First patented transgenic mouse ‘Oncomouse’ (Leder)

6. Transgenesis – the production of transgenic animals
Step 1 - DNA Preparation
Using recombinant DNA methods, build molecules of DNA containing
the gene you desire (e.g., the insulin gene)
vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
Marker gene
Termination Sequence
Promotor
Transgene
(Gene of interest)
Marker Gene
Termination Sequence
Enhancer
Step 2 – Introduction of foreign gene into the animal
A - Embryonic stem cell method
B - Pronuclear microinjection method

7. A - The Embryonic Stem Cell Method
1. Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts.
They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.
2. Transform ES cells in culture
Expose the cultured cells to the DNA so that some will incorporate it.
Microinjection
Retro virus
Electroporation
(introducing DNA from one organism into the cell of another by use of an electric pulse)
3. Transgenic stem cells are grown in vitro.
4. Select for successfully transformed cells.
5. Inject these cells into the inner cell mass (ICM) of mouse blastocysts
6. Embryo transfer
Prepare a pseudopregnant mouse
(by mating a female mouse with a vasectomized male overnight).
The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups.

8. B - The Pronucleus Microinjection Method
1. Collect eggs from a superovulated female animal
2. Firtilize eggs in vitro before the sperm head has become a pronucleus
3. Inject the male pronucleus with your transgene containing solution
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.
4. Implant the embryos in a pseudopregnant foster mother

9. A – The Pronucleus Microinjection Method
Advantages:
Most common method for creating transgenic mammalian species
Foreign DNA integrates into host cell genome at a random location
Often multiple copies of the injected DNA are incorporated at one site (concatemers)
Disadvantages:
Can’t be used for later developmental stages
Skill demanding, tedious, labour intensive, time consuming
Inefficient: about 5 % of inoculated eggs develop into transgenic animals
66 % of eggs survive the injection procedure
25 % of the implanted eggs develop into pups
25 % of the pups are transgenic
from 1000 inoculated fertilized eggs, 30 – 50 transgenic pups are produced
Not all transgenic pups will have the appropriate characteristics
(Position effect: Transgene might not be expressed because of the site of integration)

10. Transgenesis – Step 2 A B

11. Transgenesis Step 3 - Genotyping of offspring
Remove a small piece of tissue from the tail and examine its DNA for the desired gene.
Transgenic progenies are screened by PCR to examine the site of incorporation of the gene
Some transgenes may not be expressed if integrated into a transcriptionally inactive site.
No more than 10–20% will have it, and they will be heterozygous for the gene.
Step 4 - Establish a homozygous transgenic strain
Mate two heterozygous mice and screen their offspring for the 1 in 4 that will be homozygous for the transgene.

12. Examples for transgenic animals in lung research

13. Examples for transgenic animals in lung research

14. Examples – Transgenic Fish
Superfish
Increased growth and size
Growth hormone gene inserted into fertilized egg.
Transgenic salmon grows about 10 – 11 times faster than normal fish
Glo fish
freshwater zebra fish (Danio rerio)
Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish

15. Examples – Transgenic Cattle and Goats
Transgenic cows are made to produce proteins lactoferrin and interferons in their milk.
Prion free cows resistant to mad cow disease.
Goats
2009 the US FDA granted marketing approval for the first drug to be produced in genetically modified livestock.
The drug is called ATryn, which is antithrombin protein (plasminogen activator), purified from the milk of genetically modified goats.
Goats that produce spider silk etc. in their milk

16. Examples – Transgenic monkey ANDi
ANDi was the first transgenic monkey, born in 2000.
“ANDi” stands for “inserted DNA” spelled backwards.
An engineered virus was used to insert the harmless gene for green fluorescence protein (GFP) into ANDi’s rhesus genome.
ANDi proves that transgenic primates can be created, and can express a foreign gene delivered into their genome.

17. Examples – Transgenic Rabbit
Alba, the EGFP (Enhanced Green Flurescent protein) bunny
Created in 2000 as transgenic artwork

18. Ethical concerns Is it right to produce transgenic animals for:
Research?
Production of therapeutics?
Art?
If yes, even though animals are suffering (unwanted disease symptoms)?
Are there any environmental risks?
Blurring the lines between species by creating transgenic combinations.
There may be health risks associated with transgenics.
There may be long term effects on the environment when transgenic animals are released into the field.
Various bioethicist argue that it is wrong to create animals that would suffer as a result of genetic alteration.

19. Thank you!