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)

GFP transgenic mouse (Nagy) 9.5 day embryos - GFP and wt Tail tip

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

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 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!