1. Transgenic animals
Hoza, A.S
BLS 209 Lecture

2. What is a transgenic animal?
• An animal contains a foreign gene (genes)
introduced purposely by human intervention
•Transgenic animals are altered so that their DNA
produce proteins that normally they would not
produce

3. History of transgenic animal production
1970's, first transgenic mice via viral
infection, but not germline transmission
1980's, first transgenic mice via microinjection,
the most popular technique
1985, first transgenic rabbits, sheep, pigs and
cattle
80-90, commercial transgenic services, via
transgenic facility
1990's, transgenic farm animal companies as
bioreactors and organ donors

4. Different ways to create transgenic animals
Target gene – transgene
Promoter/enhancer - when, where, how much
Coding sequence - coding the specific protein
ploy A detail - mRNA stability
Transgene Structure

5. Gene transfer methods
1. Microinjection of recombinant DNA into the male
pronucleus of an in vitro fertilized egg.
2. Embryonic stem cell transfer (ES).
Other methods:
1. Chemical or
2. Viral delivery into ES cells, or homologous
recombination with ES cells.

6. Microinjection
• Inject DNA molecules (transgenes) directly into
male pronucleus
• Most popular technology, commercial available
• Success rates range from 10-30% depending on
skills and constructs
•Efficiency is not related to the copies of transgenes
injected

7. Microinjection
• The technique can be applied to other species
• No theoretical limit for the size of the construct
• Overall efficiency is still low, particularly for farm
animals
• Tandem repeat of gene constructs (head-tail)
• High frequency of mosaic
• Initial investment is high

10. Virus mediated gene transfer
• Earliest method for successful gene transfer in
mammals
• Virus has transfection property
• Killed virus is replication defective
• The virus gene is replaced with transgene gene
• The transgene is delivered to the host cell by
transfection (gene therapy)
• Can be used to transfect a wide range of cells, e.g.,
ES cells

11. Virus mediated gene transfer
•Direct transfection of embryos has resulted
nongermline transgenics
• ES cells transfection has resulted in germline
transgenics
• Has succeeded in chickens and fish
• Transfecting oocytes resulted in 100% transgenics
• Only small transgene construct is usable (8 kb or
less)
• More research is needed on the safety of the
method

12. Embryonic stem cells
•Used mostly when trying to target a transgene to
a specific site in the genome.
• Derived from ICM of blastocyst stage embryo
• Divide in vitro indefinitely without differentiation
• Contribute to development of the fetus in any
tissues, organs (germline)
• Has the potential to give rise to all tissues

13. ES cells
• May be transfected with transgene or with
genes removed (knockout) or inserted prior to
microinjection
• Has revolutionized genetics, development,
immunology and cancer research in mice

14. Steps for ES Cell Manipulation.

15. Approaches to using ES cells to create transgenic
animals.
1. The transgene can be microinjected into the
ES cells
2. can be introduced by a virus,
3. Chemical (e.g calcium phosphate or rubidium
chloride
4. by using homologous recombination.

17. Nuclear transfer
• Creation of Dolly
• Somatic cells be transfected, or genetically altered
prior to NT
• 100% efficiency of any progeny
• Low efficiency
• Abnormal development

19. Screening for Transgenic Positives
• Identification of transgene integration - DNA
• Detect transgene transcription - mRNA
• Detect transgene expression - protein

20. TRANSGENIC ANIMAL CLASSIFICATION

21. Five major categories:
1. disease models
2. transpharmers
3. xenoplanters
4. food sources
5. scientific models
1.Disease models:
animals that have been modified to exhibit the
symptoms and progression of a particular disease,
so that treatments for that disease can be tested
on them (e.g oncomouse, AIDS mouse etc)

22. 2.Transpharmers:
animals modified to express a particular
protein or suite of proteins in their milk to avoid
animal sacrifice when obtaining the drug.
The proteins can be purified to produce
medicines and hormones to treat humans, or
can possibly be administered as medicinal milk
itself.

23. Mice- commonly used to test the transpharming
transgene first.
The transgenic procedure is promising, but very
expensive, and still has a low success rate especially
for larger farm animals.
A mouse engineered in 1987 to express the clot
dissolver drug tissue plasminogen activator (tPA)
In 1990, human alphaantitrypsin, an inhibitor used to
treat emphysema, was produced in the mouse’s milk.
1997 at the New Technology Institute- human alpha-
lactalbumin in mouse’s milk

24. Larger animals like sheep, goats, and cows are the targets
for large-scale transpharming.
E.g 6 transgenic lambs for Roslin Institute - created in
1997 to produce a human clotting factor in their milk.
 The first transpharmer goats were produced in 1991 at
the Tufts University School of Veterinary Medicine to
produce tissue plasminogen activator, a clotdissolving
drug.
 transpharmer goats were produced in 1999 using
SCNT contained high levels of human antithrombin
III.

25. The first transgenic cow (Gen Pharm Intern, California),
dubbed “Herman”, and his first transgenic offspring were
bred at Gen Pharm’s lab (Netherlands)
Two calves were produced by microinjection of DNA into
embryos that were then implanted in surrogate mothers and
born alive.
One of these cows was female the transgene
rearranged itself so that a portion of the lactoferrin cDNA was
deleted.
The other calf was male, later called “Herman.” He and his
offspring contained the correctly arranged gene for human
lactoferrin.

28. 3. Xenoplanters:
animals that have been engineered to not
express the foreign antigens that normally
prevent the transplantation of their organs into
humans.
4. Food sources:
 animals that grow bigger or faster to produce
more food in a shorter amount of time with fewer
resources. E.g superpig, superfish

29. 5. Scientific models:
animals producing more or less of a
particular protein than usual,
Study that protein’s purpose in biological
mechanisms or development applied to
humans. E.g ANDi first transgenic monkey

30. TRANSGENIC ETHICS
1. Animal Rights Versus Animal Welfare
2. Right to meddle in the genomes of living beings
Transgenesis- a logical step beyond selective
breeding,
open doors past what we previously have
known to cure diseases!!??
possibly end world hunger entirely!!!??
Transgenic Art - Creating monsters!!! E.g “Alba,” the
rabbit that glows under UV light!!!!???

31. Eduardo “transgenic art.” refers to animals and plants with a
planned genome intended to express an artistic idea
symbolized by the proteins they code for.

32. 4. Animal Death Versus Human Lives Saved
low success rate in creating transgenic animals.
5. Transgenic Animals and the Environment
decrease of genetic variability within that species
Transgenic animals are not “more fit” than their “normal”
cousins.
6. Transgenic Oversight
Transgenic experimentation should be as
humane as possible.
7. Religions and Transgenic Ethics

33. Applications of transgenic farm animals
• Agricultural applications
• Bioreactors
• Organ/cell/tissue donors
• Basic research/disease model