chromosome manipulations
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A N I M A L B I O T E C H N O L O G Y
Chromosome Manipulations
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
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
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
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
Tissue culture
Animal
model
Human trials
if
successful
if
successful
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.
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.
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.
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
Matching animal systems as models for the human system
Lung and cardiovascular
Immune system
HIV and AIDS research
Dog
Mice
Monkey and chimpanzee
System Best animal model
for human
A model organism is a non-human species that isextensively studied to understand particularbiological phenomena
Enviro Pig TM
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.
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transgenic fish
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Laboratory ratA 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.
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Zebrafish
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
Easy to follow
drug effect on
embryo
development
under
microscope, sinc
e egg can
mature outside
female.
Homology Testing
Oxford Grid
Dots represent similar genes
Boxes with more than one dot
represent conserved sequences
human
Exchanging genes between individuals
Somatic cell of human
Chromosome 5
Targeted gene disruption or insertion
Homologous
Recombination
(rare event)
Cloned in tissue culture
Select for recombinant
before somatic cells
stop dividing
Reconstructed
embryo
Look for effect
of gene disruption
or insertion on
organ development
Homologous Recombination
flawed gene
good gene
Person 1
chromosome
Person 2
chromosome
Exchange section of DNA on one chromosome with
a section of DNA containing good gene on another
chromosome.
gccatt ccgtc
cggtaa ggcag
gccatt ccgtc
cggtaa ggcag
Offspring now has a copy of good gene from Person 2 in
allele donated from Person 1
Mix chromosomes
and promote DNA
replication by
mitosis.
Reconstructed embryo
Nucleus from
somatic cell
Functional
tissue or organ
Egg divides to produce
differentiated cells
An new clone, a genetic
copy of the donor, forms
when the egg starts to
divide
Step 2: insert nucleus from transformed cellGenetically
modified
somatic cells
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
Nuclear Transfer
Remove the nucleus from an egg
Suction
to hold eggPerforate egg with
needle and withdraw
intact nucleus
egg
Step 1:
C R E A T I N G D O L L Y : A B R E A K T H R O U G H I N C L O N I N G
Cloning
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?
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
Successfully cloned species
Sheep
goat
pig
cow
endangered cow (gaur)
house cat
gaur
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?
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
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
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
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
embryo
cell
nucleus
retrovirus
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
Embryonic stem cell method
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”
Transformed cells are injected into the inner cell mass of the host blastocyte for differentiation and development
blastocyte
Foreign DNA
Transformed
cell
blastocyte
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.
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
.
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.
Homologous recombination within target gene
normal gene
Useless DNA
Chromosome
with normal
gene
Plasmid with
useless DNA
insert section of DNA of gene on vector into
a section of DNA containing good gene on
chromosome of stem cells.
gccatt ccgtc
cggtaa ggcag
gccatt ccgtc
cggtaa ggcag
Chromosome is modified with a useless form of the gene. Look for a trait that has
changed
Recombination
between vector
and chromosome
Random insertion of useless gene at a location other than the target gene
normal gene
Useless DNA
Chromosome
with normal
gene
Vector with
useless DNA
Insert section of DNA of useless gene on vector into
a section of chromosome that does not disrupt target gene.
gccatt ccgtc
cggtaa ggcag
gccatt ccgtc
cggtaa ggcag
Chromosome is modified with a useless form of the gene at some other site than target
gene
Recombination
between vector
and chromosome
BlastocyteTransformed stem cell
Knockout
mouse with
nonfunctional
gene in all its
differentiated
somatic cells
chimera
Not all cells had the
trait changed
Need to crossbreed
for 2 generations to
get all cells to lose
trait.
Producing human antibodies in animals
Antibodies are proteins whose structure gives it the ability to bind very specifically to other proteins
Antibody (Ab)
Antigen
(Ag)
Region of antigen
protein that is specifically
recognized and bound by
antibody
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
Making cells that produce
monoclonal antibodies
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
The specific antibody is
released into the culture
medium and recovered
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