transgenic mice: generation and husbandry. transgenic vs. knock-out w transgenic: an organism that...
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Transgenic mice: generation and husbandry
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
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
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)
Tail tip9.5 day embryos - GFP and wt
GFP transgenic mouse (Nagy)
GFP transgenic mouse (Nagy)
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
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
Pronuclear injection
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
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
Vasectomizing
1 2
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
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
Typical KO vector
*tk:thymidine kinase
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 cellcontribution
Chimeric founder - weaker ES cellcontribution
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!