Transgenic mice generation and husbandry
Download
1 / 60

Transgenic mice: generation and husbandry - PowerPoint PPT Presentation


  • 287 Views
  • Updated On :

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

Related searches for Transgenic mice: generation and husbandry

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Transgenic mice: generation and husbandry' - Jimmy


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

Transgenic vs knock out l.jpg
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 out3 l.jpg
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 out4 l.jpg
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 l.jpg
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



Slide8 l.jpg

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 l.jpg
GFP transgenic mouse (Nagy) expressed in neuronal progenitors (neurogenin 1)

9.5 day embryos -

GFP and wt

Tail tip


Gfp transgenic mouse nagy10 l.jpg
GFP transgenic mouse (Nagy) expressed in neuronal progenitors (neurogenin 1)


Planning a transgenic production mouse colony l.jpg
Planning a Transgenic production mouse colony expressed in neuronal progenitors (neurogenin 1)

  • Mouse strain - popular

  • Colony size

    • typical injection 200 embryos (7-10 females s.o.)

    • Superovulation efficiency

    • Parenting suitability

    • Pseudo-pregs


Injecting fertilized eggs l.jpg
Injecting fertilized eggs expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Pronuclear injection expressed in neuronal progenitors (neurogenin 1)


Implantation of 1 or 2 cell embryos l.jpg
Implantation of 1 or 2 cell embryos expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Implanting 1(or 2) cell embryos expressed in neuronal progenitors (neurogenin 1)

2

1


Implanting 1 or 2 cell embryos cont l.jpg
Implanting 1(or 2) cell embryos (cont.) expressed in neuronal progenitors (neurogenin 1)

3


Pseudopregnant females and vasectomized males l.jpg
Pseudopregnant females and vasectomized males expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Vasectomizing expressed in neuronal progenitors (neurogenin 1)

1

2


Breeding tg founders l.jpg
Breeding Tg founders expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Transgenic mice as tools expressed in neuronal progenitors (neurogenin 1)

  • 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 tools23 l.jpg
Transgenic mice as tools expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Vector design expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Typical KO vector expressed in neuronal progenitors (neurogenin 1)

*tk:thymidine kinase


Embryonic stem cells l.jpg
Embryonic stem cells expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
ES cells growing in culture expressed in neuronal progenitors (neurogenin 1)


Es cells are transformed l.jpg
ES cells are transformed expressed in neuronal progenitors (neurogenin 1)

  • 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 l.jpg
Implantation of blastocysts 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 l.jpg
Implanting blastocysts blastocysts

1

2



Littermates l.jpg
Littermates blastocysts

Black mouse -

no apparent ES cell

contribution

Chimeric founder -

strong ES cell

contribution

Chimeric founder -

weaker ES cell

contribution


Chimeric mouse l.jpg
Chimeric mouse blastocysts


Testing the offspring l.jpg
Testing the offspring blastocysts

  • 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 l.jpg
Breeding Chimeras (knock-out founder) blastocysts

  • 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 l.jpg
Breeding Chimeras (knock-out founder)cont blastocysts

  • 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 l.jpg
Knock-out mice as tools blastocysts

  • 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 l.jpg
Breeding Transgenics blastocysts

  • 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 l.jpg
Breeding Transgenics (cont.) blastocysts

  • 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 cont44 l.jpg
    Breeding Transgenics (cont) blastocysts

    • 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 l.jpg
    Housing blastocysts

    • Range from conventional to barrier

    • Researcher can usually advise on level of protection that is appropriate


    Health monitoring programs l.jpg
    Health Monitoring Programs blastocysts

    • 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 programs47 l.jpg
    Health Monitoring Programs blastocysts

    • Months of research data may have to be thrown out because of undetected infection

      • Unfit for research

      • Data unreliable


    Pathogens l.jpg
    Pathogens blastocysts

    • 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 l.jpg
    Pathogens (cont): blastocystsSome 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 effects50 l.jpg
    Pathogens (cont): blastocystsSome 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 effects51 l.jpg
    Pathogens (cont): blastocystsSome 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 effects52 l.jpg
    Pathogens (cont): blastocystsSome 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 effects53 l.jpg
    Pathogens (cont.): blastocystsSome 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 effects54 l.jpg
    Pathogens (cont.): blastocystsSome 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 effects55 l.jpg
    Pathogens (cont.): blastocystsSome common pathogens and their effects

    • Reported effects: confounds carcinogenicity research; gastointestinal system research


    Pathogens cont some common pathogens and their effects56 l.jpg
    Pathogens (cont.): blastocystsSome 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 effects57 l.jpg
    Pathogens (cont.): blastocystsSome 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 effects58 l.jpg
    Pathogens (cont.): blastocystsSome 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 effects59 l.jpg
    Pathogens (cont.): blastocystsSome 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