2. Benefits of Genetically Engineered Animals
Used to develop new medical treatments
Improve our food supply
Enhance our understanding of biology of all
animals, including humans
3. Animal Models
Animal systems are a model for the human system
Polio vaccine was developed using animals as test systems
Cataract surgical procedures were developed with animals
Dialysis was tested first in animals before being applied to
human conditions
4. Regulation of animal research
Animal Welfare Act
Sets specific regulations regarding, housing, feeding, cleanliness
and medical care of animals
Researchers must first develop a plan describing
Appropriateness of species to be used
Minimum number of animals needed for test
Oversight committee reviews and approves plan
Government agencies monitor welfare of the test animals
5. Phase Testing
Testing a new product for safety in humans involves
vigorously following scientific methodology developed
for animal systems
Involves collecting data from a statistically significant number of
trials (experiments) in lab cell tissue cultures, in live animals and
in human subjects.
3-stages of testing
Human trials
Animal
model
Tissue culture
if
successful
if
successful
6. Testing
If test results using cell cultures indicates toxicity of
product, then product will never be tested on live
animals.
Testing on live animals requires evaluation of more
than one species, since different species may respond
differently.
7. Phase Testing
Animal models can provide the following
information on a new product
Absorption of chemical by body
Body metabolism of chemical
Time require for chemical or product to be excreted
If significant problems are encountered with
product in live animals, then product is never
tested in humans.
8. Side-effects of new drugs discovered in animal
models
Example
Propecia
Used
to encourage hair growth
Animal studies indicated that serious birth
defects occurred in male offspring when
pregnant animals were given large doses of
drug
As a result of animal tests, warnings were put
on containers of Propecia to avoid birth defects
in humans using drug.
9. How do you select appropriate animal as a model for the
human system?
Look for genetic homology between animal and
human systems.
In addition, identify animal that
Has short time between generations
Can produce lots of offspring in each generation
Can be easily maintained and manipulated in the laboratory
10. Matching animal systems as models for the human
system
System
Lung and cardiovascular
Best animal model
for human
Dog
Immune system
HIV and AIDS research
Mice
Monkey and chimpanzee
11. A model organism is a non-human species that is
extensively studied to understand particular
biological phenomena
12. Enviro Pig TM
12
Transgenic pigs express phytase in their salivary glands
Phytic acid in the pig meal is degraded releasing phosphorus
The phosphorus is absorbed by the pig.
Normally the phytic acid/phosphorus complex passes through the pig and is excreted as
waste
Pig waste is a major pollutant & can cause eutrophication of lakes & streams.
YANAMALA VIJAY RAJ
1/20/2014
16. Laboratory rat
A laboratory rat is a rat of the species Rattus norvegicus (brown rat) which is bred and kept
for scientific research. Laboratory rats have served as an important animal model for
research in psychology,medicine, and other fields.
16
YANAMALA VIJAY RAJ
1/20/2014
21. Zebrafish
Lots of genetic similarity to humans
Egg lends itself to genetic transfer
no need to implant an egg inside a donor mother for gestation.
Embryos are transparent, making it possible to study
cell division under microscope from first hour of
creation.
transplant gene into embryo
Because the genetics of zebrafish and humans are
similar, they are ideal animal systems for determining
whether a new drug induces genetic mutations
22. Easy to follow
drug effect on
embryo
development
under
microscope, sinc
e egg can
mature outside
female.
24. Exchanging genes between individuals
Select for recombinant
before somatic cells
stop dividing
Somatic cell of human
Cloned in tissue culture
Chromosome 5
Homologous
Recombination
(rare event)
Targeted gene disruption or insertion
Reconstructed
embryo
Look for effect
of gene disruption
or insertion on
organ development
25. Homologous Recombination
flawed gene
Person 1
chromosome
good gene
Person 2
chromosome
Mix chromosomes
and promote DNA
replication by
mitosis.
gccatt ccgtc
cggtaa ggcag
gccatt ccgtc
cggtaa ggcag
Exchange section of DNA on one chromosome with
a section of DNA containing good gene on another
chromosome.
Offspring now has a copy of good gene from Person 2 in
allele donated from Person 1
26. Reconstructed embryo
Step 2: insert nucleus from transformed cell
Egg divides to produce
differentiated cells
Genetically
modified
somatic cells
Nucleus from
somatic cell
An new clone, a genetic
copy of the donor, forms
when the egg starts to
divide
Functional
tissue or organ
27. Embryo Reconstruction
EMBRYO RECONSTRUCTION BY
TRANSPLANTATION OF THE DONOR
INNER CELL MASS TO THE RECIPIENT
BOVINE BLASTOCYST
Embryo reconstruction for chimera production
has been used in experiments oriented towards
animal science such as the production of
interspecies pregnancies in domestic animals . So
far, chimeras have been obtained by the
aggregation of the blastomeric or by inner cell
mass transplantation . The aggregation of cell
from embryos results in embryos with a more
randomly distribution contribution of cells from
each donor to the trophoblast and ICM. in their
production of interspecific sheep-goat chimeras
produced one kid by the injection of a goat ICM
into a sheep blastocyst and one lamb from the
reciprocal injection
28. Nuclear Transfer
Step 1:
Remove the nucleus from an egg
egg
Suction
to hold egg
Perforate egg with
needle and withdraw
intact nucleus
30. Embryo twinning (conventional approach)
splitting embryos in half to produce artificially created twins
commonly practiced in cattle industry today
limitation is that organisms being copied is unknown
you
may or may not end up with an animal that has
the desired characteristics and you have to wait until
the animals is full-grown to find out.
Dolly was created from an adult cell-not an embryo
Dolly was an exact copy of an adult with known
characteristics.
How is this done?
31. Cells collected from donor animal and put in a
culture medium that keeps them alive but prevents
their replication and stops gene
expression.
Egg of an animal has it’s nucleus (DNA) removed
(enucleation)
Nucleus of cultured somatic cells from donor
animal are then inserted into a recipient animal’s
egg next to its cytoplasm.
Apply low-level electric charge and fuses with egg
cytoplasm to produce a 1-cell cloned embryo.
New cell containing egg behaves as if it were an
embryonic cell rather than an adult cell. Cell
division occurs just as it would in an ordinary
fertilized egg.
Transfer embryo to surrogate mother for gestation.
Newborn will be genetically identical to donor
34. Limits of cloning
Viable cell is required
Success rate is still low
Dolly was successful only after 277 failed attempts
only 29 implanted embryos lived longer than 6 days
Many clones are born with defects
kidney problems
diabetes
crippling disabilities
old before their time-telomere length
Dolly was diagnosed with arthritis -premature aging?
35. Cloning as a means of producing
replacement body parts?
Idea is to reduce chance of cloned tissue from
being rejected by original “parent”.
It would take years for clone to produce the organs
to be used for transplant
36. Benefits of Cloning
Reduce variability of responses of a population
being used to test new drugs, etc.
avoids confounding factor of different genetic
predispositions
Preservation of endangered species
cloning pandas using common black bear as surrogate host.
Reduce time to produce new breeds of farm
animals
from 6-9 years
3 years
37. Early experiments on transgenic animals
A new gene was added to a cell grown in a tissue
culture and the effects on that one cell were
observed.
With the introduction of cloning, a gene could be
added to many cells, and all the cells could be
screened to see which one(s) contained the gene.
Each cell that contained the gene could then be used to grow
a complete animal using cloning technology
38. Transgenic techniques
Retrovirus-mediated transgenesis
infect mouse embryo with retroviruses before the embryos
are implanted into an animal for gestation.
Retrovirus acts as a vector for the new DNA
size of new DNA is limited
viruses genetic material can interfere with embryo development
not very efficient
cell
nucleus
embryo
retrovirus
39. Pronuclear injection
Introduction of foreign DNA
at earliest possible stage of
development of the zygote
(fertilized egg)
Just before the egg and
sperm cells join, DNA is
injected into the nucleus of
either cell.
Since the DNA is injected
with a syringe, no vector is
required and no vector
genetic material is
introduced that could
complicate outcome
40. Embryonic stem cell method
blastocyte
Embryonic stem cells are collected
from inner cell mass of blastocytes
Cells are mixed with foreign DNA
some cells take up the foreign DNA and
incorporate it into cell’s own DNA in the
nucleus and are “transformed”
Foreign DNA
Transformed cells are injected into
the inner cell mass of the host
blastocyte for differentiation and
development
Transformed
cell
blastocyte
41. Transgenics to make milk
healthier for humans
Lactoferrin-protein that binds iron
needed by human babies for
development
introduce gene for this protein into cells
of cow that are responsible for milk
production
Human immune genes introduced
into cows as a factory for human
antibody production.
42. Transgenics as a means of deleting genes and their
functions
Deleting a gene is a way of determining what its
function is in the cell
Active gene is replaced with a gene that has no
functional information
When the gene is “knocked out” by the useless
DNA, the trait controlled by the active gene is
eliminated from the animal
.
43. Knockout Mice
Knockout mice begin as embryonic stem cells with specifically modified
DNA that has been prepared by recombinant techniques. The
modification results in a nonsense mutation in the normal gene of the
animal.
44.
45.
46. Homologous recombination within target gene
Chromosome
with normal
gene
Plasmid with
useless DNA
normal gene
Useless DNA
gccatt ccgtc
cggtaa ggcag
Recombination
between vector
gccatt ccgtc
cggtaa ggcag
and chromosome
insert section of DNA of gene on vector into
a section of DNA containing good gene on
chromosome of stem cells.
Chromosome is modified with a useless form of the gene. Look for a trait that has
changed
47.
48. Random insertion of useless gene at a location
other than the target gene
Chromosome
with normal
gene
Vector with
useless DNA
normal gene
Useless DNA
Recombination
between vector
and chromosome
gccatt ccgtc
cggtaa ggcag
gccatt ccgtc
cggtaa ggcag
Insert section of DNA of useless gene on vector into
a section of chromosome that does not disrupt target gene.
Chromosome is modified with a useless form of the gene at some other site than target
gene
49.
50. Transformed stem cell
Knockout
mouse with
nonfunctional
gene in all its
differentiated
somatic cells
Blastocyte
chimera
Not all cells had the
trait changed
Need to crossbreed
for 2 generations to
get all cells to lose
trait.
51. Producing human antibodies in animals
Antibodies are proteins whose structure gives it
the ability to bind very specifically to other
proteins
Region of antigen
protein that is specifically
recognized and bound by
antibody
Antigen
(Ag)
Antibody (Ab)
52. Antibodies could be designed that target and
inactivate cancer cells in our bodies.
Myelomas: antibody-secreting tumors
Monoclonal Abs (mAb) are produced from
myeloma cells that produce an Ab that reacts with
only one region of an antigenic protein
53. Making cells that produce
monoclonal antibodies
The specific antibody is
released into the culture
medium and recovered
Once a cell line is identified
that produces an antibody
against a specific antigen, it
can be replicated and the cells
frozen until needed to make
the specific antibody
54. Review
Approaches to change genomes of animals
Nuclear transfer of genetically modified somatic cell into an egg.
Rapid growth of organs for transplant into donor animal.
Nuclear transfer of somatic cell into egg
implant into
surrogate to produce viable organism (Dolly)
Retrovirus mediated genetic modification in animal genome.
Nuclear transfer of embryonic stem cell into egg.
Implant into surrogate to produce viable
organism
