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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: 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
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
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 tip
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
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
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
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
Drug testing
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
Typical KO vector
*tk:thymidine kinase
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
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
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
Littermates
Black mouse no apparent ES cell contribution Chimeric founder strong ES cell contribution Chimeric founder weaker ES cell contribution
Chimeric mouse
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
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
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
Common problems:
female not good mother, check for milk - give auntie male cannibalizing litter fighting (separate) Do not reunite males
Housing
Range from conventional to barrier Researcher can usually advise on level of protection that is appropriate
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
Months of research data may have to be thrown out because of undetected infection
Unfit for research Data unreliable
Pathogens
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
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
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
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
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
Acariasis (mites)
Hairless mice not susceptible Transmission - direct contact Eradication very labour-intensive