Transcript Transgenic mice: generation and husbandry

Transgenic animals and
knockout animals
3 main ways to do biological research:
1. Do research in test tubes.
2. Do research with cells.
3. Do research directly with animals.
Transgenic animals and knockout animals
Part 1: Transgenic animals:
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Introduction to transgenic animals.
How to make transgenic animals?
How to make conditional transgenic animals?
Applications of transgenic animals.
Part 2: Knockout animals
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•
•
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Introduction to knockout animals.
How to make knockout animals?
How to make conditional knockout animals?
Applications of knockout animals.
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
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.
ES cell transformation
Injection of gene into fertilized egg
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
(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
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.
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.
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.
Conditional Transgenic mouse
The expression of transgene in transgenic mouse can be induced
Important Considerations for
Conditional Transgenes
• Transgenes have low or no expression when
not induced
• Large difference between induced and noninduced gene expression
• Transgene expression rapidly turns on or off.
• Inducer (doxycycline, tamoxifen, cre) is not
toxic and easily administered
Tetracycline Controlled
Transactivator tTA
“Tet-off”
tetR
Doxycycline blocks
tTA DNA binding
VP16
tTA binds to tetO to
activate transcription
Reverse Tetracycline Controlled
Transactivator tTA
“Tet-on”
rtetR
Doxycycline allows
rtTA to bind to tetO
VP16
Without doxcycline rtTA
can not bind to tetO
Tetracycline Regulation:
Summary
No Doxycycline
Doxycycline
tTA
expressed
not expressed
rtTA
not expressed expressed
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.
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
Example 1: Transgenic Cattle
• Cloned transgenic cattle produce milk with
higher levels of beta-caein and k-casein
Published in Nature, Jan, 2003
Example 2: Transgenic Mouse
The growth hormone gene has been engineered to be expressed
at high levels in animals.
Metallothionein
promoter
regulated by
heavy metals
The result: BIG ANIMALS
Mice fed with heavy metals are 2-3 times lar
Example 3: Transgenic Mouse
Trangenic mouse embryo in which the promoter for a gene expressed in
neuronal progenitors (neurogenin 1) drives expression of a betagalactosidase reporter gene. Neural structures expressing the reporter
transgene are dark blue-green.
Example 4: GFP transgenic mouse (Nagy)
9.5 day embryos GFP and wt
Tail tip
GFP transgenic mouse (Nagy)
Example 5: Wild and domestic trout respond differently
to overproduction of growth hormone.
So, GH is not effective to domestic trout.
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
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.
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.
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.
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
KNOCKOUT MICE
Normal (+) gene X
Isolate gene X
and insert it into vector.
Genome
Defective
(-)
Gene X
VECTOR
MARKER GENE e.g.(NeoR)
Inactivate the gene
by inserting a marker gene
that make cell resistant
to antibiotic (e.g. Neomycin)
Transfer vector
with (-) gene X
into ES cells
(embryonic stem cells)
Vector and
genome
will recombine
via homologous
sequences
Genomic gene
Homologous recombination
and gene disrution
Grow ES cells in
antibiotic containing media;
Only cell with marker gene
(without normal target gene)
will survive
Problems with homologous
recombination
Unwanted random non-homologous recombination
is very frequent.
This method provides no selection against it
Solution: Replacement vectors
The knock-out construct contains the 1) NeoR gene
flanked by 2) two segments of the target gene
and 3) the HSVtk gene
Part of the gene replaced with NeoR
ES cells are selected for integration of NeoR and
against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir
Replacement vectors
NeoR
HSVtk
Homologous
recombination
Linearized
replacement plasmid
Random integration
NeoR
NeoR+/ HSVtk-
NeoR+/ HSVtk+
HSVtk will convert gancyclovir into a
toxic drug and kill HSVtk+ cells
Typical KO vector
*tk:thymidine kinase
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 3:1
(Mendelian)
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.
ES cells growing in culture
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
Successfully transformed ES
cells are injected into
blastocysts
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
Implanting blastocysts
1
2
Implanting blastocysts
3
4
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
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.
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.
Littermates
Black mouse no apparent ES cell
contribution
Chimeric founder strong ES cell
contribution
Chimeric founder weaker ES cell
contribution
Chimeric mouse
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.
Conditional knock-out animals
How to make FLOXed gene
Gene of interest
TK
NeoR
loxP
loxP
loxP
loxP
Electroporate targeting vector
into ES cells, followed
by +/- selection
NeoR+/ HSVtkcells selected
Gene flanked by loxP sites (floxed)
Make mice and breed floxed allele to homozygousity.
Mate FLOXed mice with mice
carrying a Cre transgene
Marker gene
Promoter elements
Cre
IRES
GFP
intron
SV40 p(A)
Crucial element. Recombinase
would be expressed in accordance
with specificity of your promoter.
Promoter could be regulated !!!
artificailly or naturally
Conditional knock-out animals
inactivate a gene only in specific tissues
and at certain times during development and life.
Cre-lox technology
Your gene of interest
is flanked by 34 bp loxP sites (floxed).
Cre – a site-specific recombinase enzyme from the P1 phage.
Recognises a 34bp DNA sequence loxP =
Gene between loxP sites is removed
Cre
Cre
Cre
If CRE recombinase expressed
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.
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.
Health Monitoring Programs
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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
Health Monitoring Programs
• Months of research data may have to be
thrown out because of undetected
infection:
– Unfit for research
– Data unreliable
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!
Pathogens:
Some 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
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
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
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
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 Tlymphocyte markers; altered enzyme
activities, enhanced phagocytic activity of
macrophages, rejection of xenograft tumors
etc.
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
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
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
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
The End and Good bye!