Transgenic animals and knockout animals

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Transgenic animals and knockout animals
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• 3 main ways to do biological research
• Do research in test tubes.
• Do research with cells.
• Do research directly with animals.

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• Transgenic animals and knockout animals
• Part 1 Transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animals?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animals.

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Transgenic Animal

• Animal has one or more foreign genes inserted
  into chromosome DNA inside its cells
  artificially.
• After injecting foreign gene into the pronucleus
  of a fertilized egg or blastocyst, foreign gene
  is inserted in a random fashion into chromosome
  DNA
• Randomly (Foreign gene may disrupt an endogenous
  gene important for normal development, and the
  chance is about 10. )
• multiple copies

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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animal.

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ES cell transformation
Injection of gene into fertilized egg
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Method 1 ES cell transformation vs. Method 2
Injection of gene into fertilized egg
1. ES cell transformation works well in mice
only. Other transgenic animals are produced by
egg injection
2. ES cell transformation provides more control
of the integration step (selection of stably
transfected ES cells)
3. Injection of gene into fertilized egg is less
reliable (viability of eggs, frequency of
integration), but it helps to avoids chimeric
animals
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Injecting fertilized eggs

• The eggs are harvested from mice (superovulated
  or natural matings).
• The DNA is usually injected into the male
  pronucleus.
• The eggs can be transferred in the same day (1
  cell) or the next day (2-cells) into
  pseudopregnant female oviducts.

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Breeding Transgenic animals (transgenic founders)

• Transgenic animals Individually are backcrossed
  to non-transgenic animals.
• DO NOT intercross different founders. Each
  founder results from a separate RANDOM transgene
  integration event.

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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animals?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animal.

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Conditional Transgenic mouse
The expression of transgene in transgenic mouse
can be induced
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Important Considerations for Conditional
Transgenes

• Transgenes have low or no expression when not
  induced
• Large difference between induced and non-induced
  gene expression
• Transgene expression rapidly turns on or off.
• Inducer (doxycycline, tamoxifen, cre) is not
  toxic and easily administered

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Tetracycline Controlled Transactivator
tTA Tet-off
tetR
VP16
Doxycycline blocks tTA DNA binding
tTA binds to tetO to activate transcription
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Reverse Tetracycline Controlled Transactivator
tTA Tet-on
rtetR
VP16
Doxycycline allows rtTA to bind to tetO
Without doxcycline rtTA can not bind to tetO
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Tetracycline Regulation Summary

• No Doxycycline Doxycycline
• tTA expressed not expressed
• rtTA not expressed expressed

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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animal.

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Applications of Transgenic Animals

• Transgenic mice are often generated to
• 1. characterize the ability of a promoter to
  direct tissue-specific gene expression
• e.g. a promoter can be attached to a reporter
  gene such as LacZ or GFP
• 2. examine the effects of overexpressing and
  misexpressing endogenous or foreign genes at
  specific times and locations in the animals
• 3 Study gene function
• Many human diseases can be modeled by
  introducing the same mutation into the mouse.
  Intact animal provides a more complete and
  physiologically relevant picture of a transgene's
  function than in vitro testing.
• 4. Drug testing

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Example 1 Transgenic Cattle

• Cloned transgenic cattle produce milk with higher
  levels of beta-caein and k-casein
• Published in Nature, Jan, 2003

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Example 2 Transgenic Mouse
The growth hormone gene has been engineered to be
expressed at high levels in animals.
The result BIG ANIMALS
Mice fed with heavy metals are 2-3 times larger
Metallothionein promoter regulated by heavy
metals
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Example 3 Transgenic Mouse
Trangenic mouse embryo in which the promoter for
a gene expressed in neuronal progenitors
(neurogenin 1) drives expression of a
beta-galactosidase reporter gene. Neural
structures expressing the reporter transgene are
dark blue-green.
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Example 4 GFP transgenic mouse (Nagy)
9.5 day embryos - GFP and wt
Tail tip
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GFP transgenic mouse (Nagy)
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Example 5 Wild and domestic trout respond
differently to overproduction of growth hormone.
So, GH is not effective to domestic trout.
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Example 6 Transgenic mice as tools

• Normal mice can't be infected with polio virus.
  They lack the cell-surface Polio virus receptor.
  But, human has Polio virus receptor.
• Transgenic mice expressing the human gene for the
  Polio receptor can be infected by polio virus and
  even develop paralysis and other pathological
  changes characteristic of the disease in humans

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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animals.

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knock-out Animal

• One endogenous gene in an animal is changed. The
  gene can not be expressed and loses its
  functions.
• DNA is introduced first into embryonic stem (ES)
  cells.
• ES cells that have undergone homologous
  recombination are identified.
• ES cells are injected into a 4 day old mouse
  embryo a blastocyst.
• Knockout animal is derived from the blastocyst.

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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animals.

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Vector design

• Recombinant DNA methods Simple KO
• Structural gene desired (e.g. insulin gene) to be
  "knocked out" is replaced partly or completely by
  a positive selection marker to knock out the gene
  functions.
• Vector DNA to enable the molecules to be inserted
  into host DNA molecules

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KNOCKOUT MICE
Isolate gene X and insert it into vector.
Inactivate the gene by inserting a marker
gene that make cell resistant to antibiotic
(e.g. Neomycin)
Normal () gene X
Genome
Defective (-) Gene X
Transfer vector with (-) gene X into ES cells
(embryonic stem cells)
VECTOR
e.g.(NeoR)
MARKER GENE
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Vector and genome will recombine via homologous
sequences
Genomic gene
Exon 1
Exon 2
Exon 3
Exon 4
Homologous recombination and gene disrution
Grow ES cells in antibiotic containing media
Only cell with marker gene (without normal
target gene) will survive
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Problems with homologous recombination
Unwanted random non-homologous recombination is
very frequent. This method provides no selection
against it
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Replacement vectors
Gene segment 1
Gene segment 2
NeoR
HSVtk
Linearized replacement plasmid
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Typical KO vector
tkthymidine kinase
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Inject ES cells with (-) gene X into early
mouse embryo
Transfer embryos to surrogate mothers
Resulting chimaras have some cells with ()
gene X and (-) gene X.
Mate them with normal mice
It is lucky, if germline contain (-) gene X
Screen pups to find -/ and mate them
Next generation will split as 31 (Mendelian)
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Embryonic stem cells

• Harvested from the inner cell mass of mouse
  blastocysts
• Grown in culture and retain their full potential
  to produce all the cells of the mature animal,
  including its gametes.

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ES cells growing in culture
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ES cells are transformed

• Cultured ES cells are exposed to the vector
• Electroporation punched holes in the walls of the
  ES cells
• Vector in solution flows into the ES cells
• The cells that don't die are selected for
  transformation using the positive selection
  marker
• Randomly inserted vectors will be killed by
  gancyclovir

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Successfully transformed ES cells are injected
into blastocysts
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Implantation of blastocysts

• The blastocysts injected with transformed ES
  cells are left to rest for a couple of hours
• Expanded blastocysts are transferred to the
  uterine horn of a pseudopregnant female
• Max. 1/3 of transferred blastocysts will develop
  into healthy pups

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Implanting blastocysts
1
2
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Implanting blastocysts
3
4
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Testing the offspring

• A small piece of tissue - tail or ear - is
  examined for the desired gene
• 10-20 will have it and they will be heterozygous
  for the gene

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Breeding Chimeras (knock-out founder)

• Chimera - the founder
• germ-line transmission - usually the ES cells are
  derived from a 129 mouse strain (agouti or white
  colour) and the ES cells are injected into
  blastocyst derived from a C57Bl/6 mouse (black).
• The more that the ES cells contribute to the
  genome of the knockout mouse, the more the coat
  colour will be agouti. The chimera mouse is
  usually tiger striped.

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Breeding Chimeras (knock-out founder)

• Males that are 40 to 100 based on agouti coat
  colour should be bred
• Females should not be bred (low incidence of
  success).
• Breed aggressively- rotate females through male's
  cage. If the male produces more than 6 litters
  without transmitting knockout gene, the knockout
  gene will not likely go to germline and should
  not be used for more breeding.

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Littermates
Black mouse - no apparent ES cell contribution
Chimeric founder - strong ES cell contribution
Chimeric founder - weaker ES cell contribution
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Chimeric mouse
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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animal.

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Conditional knock-out animals How to make FLOXed
gene
TK
NeoR
Electroporate targeting vector into ES cells,
followed by /- selection
loxP
loxP
NeoR/ HSVtk- cells selected
loxP
loxP
Gene flanked by loxP sites (floxed)
Make mice and breed floxed allele to
homozygousity.
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Mate FLOXed mice with mice carrying a Cre
transgene
Marker gene
Promoter elements Cre IRES GFP
SV40 p(A)
intron
Crucial element. Recombinase would be expressed
in accordance with specificity of your
promoter. Promoter could be regulated
!!! artificailly or naturally
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Conditional knock-out animals
inactivate a gene only in specific tissues and
at certain times during development and life.
Your gene of interest is flanked by 34 bp loxP
sites (floxed).
If CRE recombinase expressed
Gene between loxP sites is removed
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• Transgenic animals and knockout animals
• Part 1 transgenic animals
• Introduction to transgenic animals.
• How to make transgenic animals?
• How to make conditional transgenic animal?
• Applications of transgenic animals.
• Part 2 Knockout animals
• Introduction to knockout animals.
• How to make knockout animals?
• How to make conditional knockout animals?
• Applications of knockout animal.

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Applications of Knock-out animals

• Find out if the gene is indispensable
  (suprisingly many are not!)
• Check the phenotypes of knockout animals
• Determine the functions of knockout gene.

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Health Monitoring Programs

• Costly
• Monitor health status of colony
• Long-term savings time, effort, money
• Inform investigator (collaborators) of pathogen
  status
• Prevent entry of pathogens
• Promptly detect and deal/eliminate pathogen entry

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Health Monitoring Programs

• Months of research data may have to be thrown out
  because of undetected infection
• Unfit for research
• Data unreliable

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Pathogens

• Viral, bacterial, parasitic, and fungal
• Sometimes no overt signs
• Many alter host physiology - host unsuitable for
  many experimental uses
• Cures can be bad too!

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PathogensSome common pathogens and their effects

• Sendai virus
• Mouse, rat, hamsters
• One of the most important mouse pathogens
• Transmission - contact, aerosol - very contagious
• Clinical signs - generally asymptomatic minor
  effects on reproduction and growth of pups

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Pathogens (cont)Some common pathogens and their
effects

• Infected shortly after birth
• stop breeding
• Altered physiology as the virus travels down the
  respiratory tract -necrosis of airway epithelium,
  pneumonia in lungs, lesions.
• 129/J and DBA, aged and immunodeficient mice most
  susceptible SJL/J and C57Bl/6 most resistant

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Pathogens (cont)Some common pathogens and their
effects

• Reported effects
• Interference with early embryonic development and
  fetal growth
• Alterations of macrophage, natural killer (NK)
  cell, and T- and B-cell function
• Pulmonary hypersensitivity
• Wound healing

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Pathogens (cont)Some common pathogens and their
effects

• MHV
• Probably most important pathogen of laboratory
  mice
• Extremely contagious aerosol, direct contact
• No carrier state
• Clinic state varies dependent upon MHV and mouse
  strains

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Pathogens (cont.)Some common pathogens and
their effects

• Diarrhea, poor growth, death
• Immunodeficient (e.g. nu/nu) wasting syndrome
  -eventual death
• Reported effects necrotic changes in several
  organs, including liver, lungs, spleen,
  intestine, brain, lymph nodes, and bone marrow
  differentiation of cells bearing T-lymphocyte
  markers altered enzyme activities, enhanced
  phagocytic activity of macrophages, rejection of
  xenograft tumors etc.

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Pathogens (cont.)Some common pathogens and
their effects

• Helicobacter spp
• H. Hepaticus (mice) most prominent
• Transmission direct fecal-oral
• Clinical signs absent in immunocompetent mice
• In immunodeficient mice- rectal prolapse
• Pathological changes chronic, active hepatitis,
  enterocolitis, hepatocellular neoplasms

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Pathogens (cont.)Some common pathogens and
their effects

• Oxyuriasis (Pinworms)
• Mouse pinworms (Syphacia obvelata) has been
  reported to infect humans
• Eggs excreted in faeces, can aerosolize - wide
  spread environmental contamination
• Infection rate high infection usually sub
  clinical
• Athymic (nu/nu) mice are more susceptible

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Pathogens (cont.)Some common pathogens and
their effects

• Few reports documenting the effects of pinworms
  on research, many consider irrelevant
• Acariasis (mites)
• Hairless mice not susceptible
• Transmission - direct contact
• Eradication very labour-intensive

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Pathogens (cont.)Some common pathogens and
their effects

• Reported to have caused
• altered behaviour
• selective increases in immunoglobulin G1 (IgG1),
  IgE, and IgA levels and depletion in IgM and IgG3
  levels in serum
• Lymphocytopenia
• Granulocytosis
• Increased production of IL-4 decreased
  production of IL-2

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The End and Good bye!