Transcript Transgenic mice: generation and husbandry - univ

Transgenic mice: generation and
husbandry
Transgenic vs. “knock-out”
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Transgenic: an organism that has had DNA
introduced into one or more of its cells
artificially
“transgenic”: DNA is integrated in a
random fashion by injecting it into the
pronucleus of a fertilized ovum
• Random (approx.. 10% disrupt an endogenous
gene important for normal development)
• multiple copies
Transgenic vs. “knock-out”


Transgenic: an organism that has had DNA
introduced into one or more of its cells
artificially
“transgenic”: DNA is integrated in a
random fashion by injecting it into the
pronucleus of a fertilized ovum
• Random (approx.. 10% disrupt an endogenous
gene important for normal development)
• multiple copies
Transgenic vs. “knock-out”
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“knockout”: DNA is introduced first into
embryonic stem (ES) cells. ES cells that
have undergone homologous recombination
are identified and injected into a 4 day old
mouse embryo - a blastocyst
• targeted insertion
Transgenic production
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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
Brinster's growth hormone 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. (Dr. Anne Calof)
GFP transgenic mouse (Nagy)
9.5 day embryos GFP and wt
Tail tip
GFP transgenic mouse (Nagy)
Planning a Transgenic
production mouse colony
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Mouse strain - popular
Colony size
• typical injection 200 embryos (7-10 females
s.o.)
• Superovulation efficiency
• Parenting suitability
• Pseudo-pregs
Injecting fertilized eggs
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The eggs are harvested 0.5 dpc
(superovulated or natural matings)
The DNA is usually injected into the male
pronucleus
The eggs can be transferred the same day or
the next (2-cell) into pseudopregnant female
oviducts
Pronuclear injection
Implantation of 1 or 2 cell
embryos
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The injected eggs are implanted the same
day or are incubated overnight and
implanted the next day
Injected eggs are transferred to the oviduct
of a 0.5 dpc pseudopregnant female
Implanting 1(or 2) cell embryos
1
2
Implanting 1(or 2) cell embryos
(cont.)
3
Pseudopregnant females and
vasectomized males
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Female mice can be tricked into thinking
they are pregnant
A mouse in estrus is mated with a
vasectomized male
pseudopregnancy
If eggs (blastocysts) implanted will become
truly pregnant and will give birth to live
offspring
Vasectomizing
1
2
Breeding Tg founders
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Individually backcrossed to the strain of
choice
DO NOT intercross different founders each founder results from a separate
RANDOM transgene integration even
Transgenic mice as tools
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Study gene function
• Many human diseases can be modeled by
introducing the same mutation into the mouse.
Intact organism provides a more complete and
physiologically relevant picture of a transgene's
function than in vitro testing
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Drug testing
Transgenic mice as tools
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Polio virus receptor
Normal mice can't be infected with polio
virus. They lack the cell-surface molecule
that, in humans, serves as the receptor for
the virus.
Tg mice expressing the human gene for the
receptor can be infected by polio virus and
even develop paralysis and other
pathological changes characteristic of the
disease in humans
Vector design
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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. (knock out
function!)
• Vector DNA to enable the molecules to be
inserted into host DNA molecules
Typical KO vector
*tk:thymidine kinase
Embryonic stem cells
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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
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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
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The blastocysts are left to rest for a couple
of hours
Expanded blastocysts are transferred to the
uterine horn of a 2.5 dpc pseudopregnant
female
Max. 1/3 of transferred blasts will develop
into healthy pups
Implanting blastocysts
1
2
Implanting blastocysts (cont.)
3
4
Littermates
Black mouse no apparent ES cell
contribution
Chimeric founder strong ES cell
contribution
Chimeric founder weaker ES cell
contribution
Chimeric mouse
Testing the offspring
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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)
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Chimera - the founder
• germ-line transmission - usually the ES cells
are derived from a 129 strain (agouti or white
colour) and the ES cells are injected into a
C57Bl/6 blastocyst (black). The more that the
ES cells contribute to the genome of the mouse,
the more the coat colour will be agouti. The
chimera mouse is usually “tiger” striped.
Breeding Chimeras (knock-out
founder)cont
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Males that are 40% to 100% based on
agouti coat colour should be bred
Females should not be bred (low incidence
of success) ES cells are male.
Breed aggressively- rotate females through
male's cage. If the male produces more than
6 litters without transmitting, not likely to
go germline and should be sac'ed
Knock-out mice as tools
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If the replacement gene is nonfunctional
(null allele), mating of the heterozygous
will produce a strain of "knock-outs'
homozygous for the nonfunctional gene
(both copies are knocked-out
• Find out if the gene is indispensable
(suprisingly many are not!)
• "pleiotropic" expression in different tissues in
different ways and at different times in
development
Breeding Transgenics
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Most transgenics are bred onto a C57Bl/6
background
• standard
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BL/6 breeding information
• mate 6-8 weeks for best reproductive
performance
• replace males when 1 year old
Breeding Transgenics (cont.)
• Replace females after 6 litters or at 6 months of
age
• quick breeding - 1 founder male: 2 females
• rotation of females through male cage
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Common problems:
• female not good mother, check for milk - give
auntie
• male cannibalizing litter
• fighting (separate) Do not “reunite” males
Breeding Transgenics (cont)
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Stick to schedules or be overwhelmed
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strict records (birth, ID, parents)
ID pups
tail tip or collect ear tissue at 2 weeks
try to genotype before weaning
wean only positives, sac negatives (mosaics?)
house male and females separately
mate at 6 weeks
Housing
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Range from conventional to barrier
Researcher can usually advise on level of
protection that is appropriate
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
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Months of research data may have to be
thrown out because of undetected infection
• Unfit for research
• Data unreliable
Pathogens
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Viral, bacterial, parasitic, and fungal
• Sometimes no overt signs
• Many alter host physiology - host unsuitable
for many experimental uses
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Cures can be bad too!
• Parasiticide - Ivermectin - immune systemmodulating activity
Pathogens (cont):
Some common pathogens and
their effects
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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
• No carrier state - stop breeding
• Altered physiology: as the virus travels down
the resp.. tract -necrosis of airway epithelium,
pneumonia in lungs, lesions.
• 129/J and DBA, aged and immunodeficient
most susceptible; SJL/J and C57Bl/6 most
resistant
Pathogens (cont):
Some common pathogens and
their effects
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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
• Isograft rejection
• Wound healing
Pathogens (cont):
Some common pathogens and
their effects
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MHV
• Probably most important pathogen of
laboratory mice
• Extremely contagious; aerosol, direct contact;
fomites
• 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
• Immunocompromised 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,
bilirubin concentration, enhanced phagocytic
activity of macrophages, rejection of xenograft
tumors etc. etc. etc.
Pathogens (cont.):
Some common pathogens and
their effects
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Helicobacter spp
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Genus keeps expanding with discoveries
H. Hepaticus (mice) most prominent
Transmission: direct fecal-oral or fomites
Clinical signs absent in immunocompetent
Immunodeficient - rectal prolapse
Pathological changes: chronic, active hepatitis,
enterocolitis, hepatocellular neoplasms
Pathogens (cont.):
Some common pathogens and
their effects
• Reported effects: confounds carcinogenicity
research; gastointestinal system research
Pathogens (cont.):
Some common pathogens and
their effects
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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
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Acariasis (mites)
• Hairless mice not susceptible
• Transmission - direct contact
• Eradication very labour-intensive
Pathogens (cont.):
Some common pathogens and
their effects
• C57Bl very susceptible
• Infestation: asymptomatic or may cause
wasting; scruffiness; pruritus; patchy alopecia;
accumulation of fine bran-like material, mostly
over affected areas; self-trauma to the point of
amputation; and secondary pyoderma
• Pathological changes: hyperkeratosis,
erythema, mast cell infiltration, ulcerative
dermatitis, splenic lymphoid and lymph node
hyperplasia;
Pathogens (cont.):
Some common pathogens and
their effects
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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!