Transgenic animals

1. Chapter 7
Animal Biotechnology

2. Animals in Research

3. B1
B4
B2
B3
Animals in Research

4. Animals in Research
Animation of
Drug
Development
Process

5. z Animal Models
• Mice
• Rats
• Zebrafish (3 month generation time,
200 progeny, complete embryogenesis
in 120 hrs)
• Dogs (lungs and cardiovascular
system)
• Cats
• Pigs (PPL Therapeutics- delete a gene
which causes hyperacute rejection of
pig-to-human organ transplantation)
• Primates (HIV and AIDs research,
geriatric research)
Animals in Research

6. z Alternatives to Animal Models
• Cell culture devices
• Researchers use cell cultures and computer-generated
models whenever possible, but this doesn’t work for looking
at an organ or entire animal
Animals in Research

7. z Regulation of Animal Research
• The “Three Rs”
• Reduce the number of higher species (cats, dogs,
primates) used
• Replace animals with alternative models
whenever possible
• Refine tests and experiments to ensure the most
humane conditions possible
Animals in Research

8. z Veterinary Medicine as Clinical Trials
• Treatments for humans may also be useful for
treatments with animals (e.g. the BRCA1 gene found
in 65% of human breast tumors is similar to the
BRCA1 gene in dogs)
• Hyperthermia + radiation = more effective at killing
tumors
• Stimulation of cytokines for curing skin cancers
Animals in Research

9. Bioengineering Mosquitoes to Prevent Malaria
• Cloned in a gene that prevents the parasite from
crossing the midgut; blocking the continuation of its life
cycle
• Developed an antibody that prevents the parasite from
entering the mosquito’s salivary gland
Animals in Research

10. Clones
z Cloning
• Creating Dolly
• Limits to Cloning: The donor cell must
come from a living organism
• An organism is also shaped by its
environment
• The success rate for cloning is very low
• Clones may be old before their time
• The future of cloning: preservation of
endangered animals, studying the effect
of drugs etc on duplicates, improve
agricultural production

11. Transgenic Animals
z Pronuclear microinjection
z Retrovirus-mediated transgenesis
z Embronyic stem cell method
z Sperm-mediated transfer

13. A pronucleus ( pl. : pronuclei)
denotes the nucleus found in
either a sperm or egg cell duri
the process of fertilization.

14. Retrovirus-mediated transgenesis
z In transgenic animal production, gene transfer efficiency is the limiting factor
in transgenesis success rates.
z Among many gene transfer systems developed to date, the retrovirus vector-
mediated gene transfer system has been an matchless choice in gene transfer
efficiency.
z The most important features of retroviruses in regard to their use as vectors are the
technical ease and effectiveness of gene transfer, due to their affinity and infectivity
for certain target cells, leading to successful transgene incorporation.
z Once cells are infected by retroviruses, the resultant viral DNA, after reverse
transcription and integration, becomes a part of the host cell genome and is
maintained for the life of the host cell.
z In addition, it is believed that DNase hypersensitive regions are the preferred
targets for retrovirus integration implying efficient expression of exogenous proviral
genes even though the proviral copy number for each integration site is limited to a
single copy.
z Unlike DNA microinjection, integration of a viral gene does not seem to induce
rearrangements of the host genome

15. z Improving Agricultural Products
with Transgenics
• Faster growth rates or shorter
growth patterns (improve the
product), more product
• Increase nutritional content-
lactoferrin
• Turning the animals into efficient
grazers
• Transfer antimicrobial genes to
farm animals
Transgenic Animals

16. The majority of somatic ce
are leukocytes (white blood
which become present in in
numbers in milk usually as
response to a mastitis-cau
pathogen - and a small num
epithelial cells, which are m
producing cells shed from
udder when an infection oc

17. z Transgenic Animals as Bioreactors
• Biosteel otherwise known as spider silk, cloned into
goat milk (“silkmilk” goats)
• Goats reproduce faster than cows and are cheaper than
cows
• Hens also make good bioreactors in that they are cheap
and a lot of eggs are produced at one time
Transgenic Animals

18. Transgenic Animals as
Bioreactors
Researchers from the University of Wyoming have developed a way to
incorporate spiders' silk-spinning genes into goats, allowing the researchers to
harvest the silk protein from the goats’ milk for a variety of applications.
For instance, due to its strength and elasticity, spider silk fiber could have
several medical uses, such as for making artificial ligaments and tendons, for
eye sutures, and for jaw repair.
The silk could also have applications in bulletproof vests and improved car
airbags.
z Why is the military interested in goat milk spider
silk?
z The protein's antimicrobial capabilities also allows its for wound
patch production. Safety equipment used in the military, sports, and
automotive industries can benefit from this by incorporating them
on protective gears, helmets, body and vehicle armors, cables,
bulletproof vests, tires, and car airbags

19. z Knock-outs: A Special Case of Transgenesis
• A specific gene is disrupted or removed such that it is not
expressed
• Procedure: DNA is modified, it is added to embryonic stem (ES)
cells, where it undergoes homologous recombination.
• The modified ES cells are then introduced into normal embryo.
The embryo is implanted in an incubator mother.
• The offspring is a chimera. It may take several generations of
crossbreeding are required to produce animals that are complete
knock-outs.
• Breast cancer mouse
Transgenic Animals

21. Antisense or RNA Interference Approaches to Decrease Gene
Expression
z Silencing of genes
z 1. In an antisense gene construct
delivered into the plant genome, the gene to
be downregulated (that is, silenced) is
essentially put in “backwards” into a plant
transformation vector.
z When the backwards gene is transcribed, the
messenger RNA (mRNA) produced from
the transgene interferes with the translation
of complementary mRNA of the gene to be
silenced in the plant or pest into protein

22. RNAi technology
gene silencing
z In addition to the traditional strategies for vector construction
and genetic modification strategies described,
z RNAi (interference) technology is now becoming a new
way to improve the contents and fight the diseases of crop
plants.
z The first step involves degradation of dsRNA into small
interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an
RNase III-like activity.
z In the second step, the siRNAs join an RNase complex, RISC
(RNA-induced silencing complex), which acts on the
similar mRNA and degrades it.
z In certain instances, the DNA expressing the target mRNA
also undergoes methylation as a by-product of the
degradation process

24. z The RNA-induced silencing complex,
or RISC, is a multiprotein complex,
specifically a ribonucleoprotein, which
incorporates one strand of a double-
stranded RNA (dsRNA) fragment, such as
small interfering RNA (siRNA) or
microRNA (miRNA).

25. Producing Human Antibodies in
Animals
z Production of Monoclonal
antibodies (Mabs)
• Used to treat cancer, heart
disease, and transplant
rejection
• HUMANIZED monoclonal
antibodies were developed
to prevent the human anti-
mouse antibody (HAMA)
response

26. A monoclonal antibody (mAb, more rarely called moAb) is an
antibody produced from a cell lineage made by cloning a unique
white blood cell. All subsequent antibodies derived this way trace
back to a unique parent cell.
• Multiple myeloma. Multiple myeloma cells are abnormal plasma
cells (a type of white blood cell) that build up in the bone marrow
and form tumors in many bones of the body. Normal plasma cells
make antibodies to help the body fight infection and disease.
• A hybrid cell made in the laboratory by fusing a normal cell with a
cancer cell, usually a myeloma or lymphoma, in order to combine
desired features of each, as the ability of the cancer cell to multiply
rapidly with the ability of the normal cell to dictate the production
of a specific antibody.

27. Composition and function
of HAT medium
 HAT medium contains hypoxanthine-aminopterin-thymidine.
Hypoxanthine, aminopterin, and thymidine selection (HAT) medium
is commonly prepared from two stock solutions, 100× HT and 100×
A. 1. To prepare 100 mL of 100× HT, dissolve 136 mg of
hypoxanthine and 38 mg of thymidine in 100 mL of H2O suitable for
tissue culture.
 This mechanism is also used in monoclonal antibody production.
HAT(hypoxanthine–aminopterin–thymidine) medium is used for the
selection of fused hybridoma cells in the process of monoclonal
antibody production.
 After fusion of myelomas and spleen cells, HAT medium is used