Animal Biotechnology

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Chapter 7

• Animal Biotechnology

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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

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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

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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

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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
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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.

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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.

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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.

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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

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Matching animal systems as models for the human
system
System Best animal model
for human

• Lung and cardiovascular
• Immune system
• HIV and AIDS research

• Dog
• Mice
• Monkey and chimpanzee

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Zebrafish as a model organism

• Popular hardy aquarium fish
• Size of a paperclip
• Can live in small spaces
• Spawn continuously
• 3 months between generations
• 200 progeny/week/female
• Complete organ development within 120 hours of
  birth
• Because the embryos inside a female are easily
  visible to naked eye, they are ideal animal
  systems for evaluating the effect of a new drug
  on development

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Homology Testing
Oxford Grid
human
Dots represent similar genes Boxes with more than
one dot represent conserved sequences
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Easy to follow drug effect on embryo development
under microscope, since egg can mature outside
female.
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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

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Exchanging genes between individuals
stopped
Select for recombinant before somatic cells stop
dividing
Somatic cell of human
Cloned in tissue culture
Reconstructed embryo
Chromosome 5
Homologous Recombination (rare event)
Look for effect of gene disruption or insertion
on organ development
Targeted gene disruption or insertion
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Homologous Recombination
flawed gene
Person 1 chromosome
good gene
Person 2 chromosome
gccatt ccgtc cggtaa ggcag
Mix chromosomes and promote DNA replication by
mitosis.
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
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Embryo Reconstruction
Cells generated from original somatic cell in
which homologous recombination occurred
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Nuclear Transfer
Step 1
Remove the nucleus from an egg
egg
Suction to hold egg
Perforate egg with needle and withdraw intact
nucleus
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Reconstructed embryo
Genetically modified somatic cells
Step 2 insert nucleus from transformed cell
Nucleus from somatic cell
Egg divides to produce differentiated cells
An new clone, a genetic copy of the donor, forms
when the egg starts to divide
Functional tissue or organ
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Cloning

• Creating Dolly A breakthrough in cloning

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• 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?

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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 its nucleus
(DNA) removed (enucleation) Nucleus of cultured
somatic cells from donor animal are then inserted
into a recipient animals 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
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Successfully cloned species

• Sheep
• goat
• pig
• cow
• endangered cow (gaur)
• house cat

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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?

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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

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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

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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

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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
embryo
nucleus
retrovirus
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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

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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 cells 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

Transformed cell
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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.

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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

.
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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.
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Homologous recombination within target gene
Chromosome with normal gene
normal gene
Useless DNA
Plasmid with useless DNA
gccatt ccgtc cggtaa ggcag
Recombination between vector and chromosome
gccatt ccgtc cggtaa ggcag
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
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Random insertion of useless gene at a location
other than the target gene
Chromosome with normal gene
normal gene
Useless DNA
Vector with useless DNA
gccatt ccgtc cggtaa ggcag
Recombination between vector and chromosome
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
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Transformed stem cell
Blastocyte
Knockout mouse with nonfunctional gene in all its
differentiated somatic cells
Not all cells had the trait changed
Need to crossbreed for 2 generations to get all
cells to lose trait.
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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
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• 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

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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
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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
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• Pronuclear injection