transgenic mice: generation and husbandry. transgenic vs. knock-out w transgenic: an organism that...

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!