transgenic animals 4

8/4/2019 Transgenic Animals 4

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Transgenic mice: generation and

husbandry


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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


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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


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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 andmisexpressing endogenous or foreign genes at

specific times and locations in the animals


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Brinster's growth hormone mouse


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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)


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Tail tip9.5 day embryos -

GFP and wt

GFP transgenic mouse (Nagy)


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GFP transgenic mouse (Nagy)


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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


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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 femaleoviducts


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Pronuclear injection


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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


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Implanting 1(or 2) cell embryos

1 2


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Implanting 1(or 2) cell embryos

(cont.)

3


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Pseudopregnant females and

vasectomized males Female mice can be tricked into thinking

they are pregnant

A mouse in estrus is mated with avasectomized male

pseudopregnancy

If eggs (blastocysts) implanted will becometruly pregnant and will give birth to liveoffspring


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Vasectomizing

1 2


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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


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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


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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


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Vector design

Recombinant DNA methods: Simple KO

Structural gene desired (e.g. insulin gene) to be

"knocked out" is replaced partly or completelyby a positive selection marker. (knock out

function!)

Vector DNA to enable the molecules to be

inserted into host DNA molecules


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Typical KO vector

*tk:thymidine kinase


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Embryonic stem cells

Harvested from the inner cell mass of

mouse blastocysts

Grown in culture and retain their fullpotential to produce all the cells of the

mature animal, including its gametes


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ES cells growing in culture


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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 bygancyclovir


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Successfully transformed ES cells

are injected into blastocysts


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Implantation of blastocysts

The blastocysts are left to rest for a couple

of hours

Expanded blastocysts are transferred to theuterine horn of a 2.5 dpc pseudopregnant

female

Max. 1/3 of transferred blasts will developinto healthy pups


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Implanting blastocysts

1 2


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Implanting blastocysts (cont.)

3 4


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Littermates

Black mouse -

no apparent ES cell

contribution

Chimeric founder -

strong ES cell

contribution

Chimeric founder -

weaker ES cell

contribution


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Chimeric mouse


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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 beheterozygous for the gene


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Breeding Chimeras (knock-out

founder) Chimera - the founder

germ-line transmission - usually the ES cells

are derived from a 129 strain (agouti or whitecolour) 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. Thechimera mouse is usually tiger striped.


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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 incidenceof success) ES cells are male.

Breed aggressively- rotate females throughmale's cage. If the male produces more than6 litters without transmitting, not likely togo germline and should be sac'ed


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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


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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


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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 - giveauntie

male cannibalizing litter

fighting (separate) Do not reunite males


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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


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Housing

Range from conventional to barrier

Researcher can usually advise on level of

protection that is appropriate


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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


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Health Monitoring Programs

Months of research data may have to be

thrown out because of undetected infection

Unfit for research Data unreliable


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Pathogens

Viral, bacterial, parasitic, and fungal

Sometimes no overt signs

Many alter host physiology - host unsuitablefor many experimental uses

Cures can be bad too!

Parasiticide - Ivermectin - immune system-modulating activity

P h ( )


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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

P h ( )


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Pathogens (cont):


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

P th ( t)


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Pathogens (cont):


their effects

Reported effects

Interference with early embryonic developmentand fetal growth

Alterations of macrophage, natural killer (NK)

cell, and T- and B-cell function

Pulmonary hypersensitivity

Isograft rejection

Wound healing

P th ( t)


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Pathogens (cont):


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.):


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Pathogens (cont.):


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.

P th ( t )


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Pathogens (cont.):


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

P th ( t )


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Pathogens (cont.):


their effects Reported effects: confounds carcinogenicity

research; gastointestinal system research

P th ( t )


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Pathogens (cont.):


their effects

Oxyuriasis (Pinworms)

Mouse pinworms (Syphacia obvelata) has beenreported 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 ):


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Pathogens (cont.):


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 ):


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Pathogens (cont.):


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 ):


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Pathogens (cont.):


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


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The End and Good bye!

transgenic animals 4

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