Transgenic mice
What are Transgenic Organisms?
A transgenic animal is animal whose
chromosomes have been changed to
carry the genes of other organisms.
• An organism (mice) whose
genome
• has been artificially modified
by means of genetic
engineering
• has to be transmitted
hereditary
• expression of exogenous gene
• over-expression of endogenous
gene
Brinster's growth hormone mouse
How are they created?
DNA
Microinjection
▪ Retrovirus-
mediated gene
transfer
Embryonic stem
cell-mediated gene
transfer
DNA Microinjection
Direct microinjection of a chosen gene construct from
another member of the same species or from a
different species, into the pronucleus of a fertilized
ovum.
Retrovirus-mediated gene transfer
Retroviruses are used as vectors
Infects host cells
Not all cells carry the retrovirus
The retrovirus must integrate into the germ
cells to work.
Embryonic stem cell-mediated gene
transfer
Insertion of desired DNA sequence into an in vitro
culture of embryonic stem (ES) cells.
Stem cells can give rise to a complete organism.
The cells are then incorporated into an embryo at the
blastocyst stage of development.
Transgenic production
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
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 -
Tail tip
GFP and wt
GFP transgenic mouse (Nagy)
Construct
Planning a Transgenic
production mouse colony
Mouse strain - popular
Colony size
• typical injection 200 embryos (7-10 females
s.o.)
• Superovulation efficiency
• Parenting suitability
• Pseudo-pregs
The procedures of producing transgenic mice
Preparation of Mice8
• Superovulated female mice
• to get the fertilized eggs >>
• Follicular development is stimulated with Pregnant Mare’s
Serum Gonadotrophin (PMSG)
• Ovulation is stimulated with Human Chorionic Gonadotrophin
(HCG)
• Age : 4-8 weeks
• Mate with fertile stud mice
• Check the copulation plug after mating
Steps in Production of transgenic mice
Injection of hormone PMS and hHCG
The procedures of producing transgenic
mice
Preparation of Mice8
• Fertile stud male mice
• Mate with superovulated female mice
• Age 6-8 weeks with good reproduction performance
• Sterile male mice
• has been vasectomized
• mate with pseudopregnant female mice
• Age of >> 8 weeks
Vasectomization in the Sterile Male Mice
21
Vasectomizing
1 2
Steps in Production of transgenic mice
Isolation and collecting of fertilized eggs
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Steps in Production of transgenic mice
Isolation and collecting of fertilized eggs
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Steps in Production of transgenic mice
Isolation and collecting of fertilized eggs
Wednesday, AgustEstablishing
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Steps in Production of transgenic mice
Isolation and collecting of fertilized eggs
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Steps in Production of transgenic mice
Isolation and collecting of fertilized eggs
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Steps in Production of transgenic mice
Injection of transgene into male pronuclear of
fertilized eggs
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Injecting fertilized eggs
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
Steps in Production of transgenic mice
Injection of transgene into male pronuclear of
fertilized eggs
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Steps in Production of transgenic mice
Injection of transgene
into male pronuclear
of fertilized eggs
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Pseudopregnant females and
vasectomized males
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
Steps in Production of transgenic mice
Transfer of fertilized eggs containing transgene into
oviduct of pseudopregnant female mice
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Steps in Production of transgenic mice
Transfer of fertilized eggs containing transgene into
oviduct of pseudopregnant female mice
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Steps in Production of transgenic mice
Transfer of fertilized eggs containing transgene into
oviduct of pseudopregnant female mice
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Implantation of 1 or 2 cell
embryos
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
Steps in Production of transgenic mice
Maintain the “candidate” for transgenic
mice
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Breeding Tg founders
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
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
Drug testing
Transgenic mice as tools
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
Collage
on Photostory
Dolly
Pictures to go somewhere in the
project
Genetically modified organisms are
called transgenic organisms.
TRANSGENIC
ANIMALS
1. Chickens – more
resistant to infections
2. Cows – increase milk
supply and leaner
meat
4. Goats, sheep and pigs
– produce human
proteins in their milk
Knock out Animal
Techniques in Mouse
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”
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”
“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
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. (knock out
function!)
• Vector DNA to enable the molecules to be
inserted into host DNA molecules
II. Knocking out a gene
Homologous recombination
• Clone gene that is nonfunctional
• Introduce DNA into cell by any method discussed above
• Homologous recombination will occur replacing endogenous gene
with nonfunctional gene
Homologous recombination
BMP7
Conditional Mutant: Cre-LoxP
Conditional mutants are needed when you want to study
the effects of a gene in certain tissue late in development
but the gene is also necessary early in development. A
traditional knockout would result in a mutant that does not
develop to stage needed.
Cre is a recombinase that excises DNA located in between
LoxP sites
You generate two transgenic lines one that expresses Cre
in the tissue you are interested and a second that contains
gene of interest flanked by loxP sites. The gene will only
be deleted where Cre is expressed.
• Can also activate genes: In second line place stop
signal flanked by loxP between 5’ regulatory element
and gene. When stop signal is removed gene will be
activated.
Cre / loxP Recombination
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 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
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 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
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
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
Most transgenics are bred onto a C57Bl/6
background
• standard
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
Common problems:
• female not good mother, check for milk - give
auntie
• male cannibalizing litter
• fighting (separate) Do not “reunite” males
Breeding Transgenics (cont)
Stick to schedules or be overwhelmed
• 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
Range from conventional to barrier
Researcher can usually advise on level of
protection that is appropriate
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
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!
• Parasiticide - Ivermectin - immune system-
modulating activity
Pathogens (cont):
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
• 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
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
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 T-
lymphocyte 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
Helicobacter spp
• 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
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
• 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
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!
