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Transgenic animals and knockout animals. 3 main ways to do biological research: Do research in test tubes. Do research with cells. Do research directly with animals. Transgenic animals and knockout animals Part 1: Transgenic animals: Introduction to transgenic animals.

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slide2
3 main ways to do biological research:
  • Do research in test tubes.
  • Do research with cells.
  • Do research directly with animals.
slide3
Transgenic animals and knockout animals

Part 1: Transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animals?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animals.
transgenic animal
Transgenic Animal
  • Animal hasone or more foreign genes inserted into chromosome DNA inside its cellsartificially.
  • After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst, foreign gene is inserted in a random fashion into chromosome DNA:
    • Randomly (Foreign gene may disrupt an endogenous gene important for normal development, and the chance is about 10%. )
    • multiple copies
slide5
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animal.
slide6

ES cell transformation

Injection of gene into fertilized egg

method 1 es cell transformation vs method 2 injection of gene into fertilized egg
Method 1: ES cell transformation vs. Method 2: Injection of gene into fertilized egg

1. ES cell transformation works well in mice only.

Other transgenic animals are produced by egg injection

2. ES cell transformation provides more control of the integration step

(selection of stably transfected ES cells)

3. Injection of gene into fertilized egg is less reliable

(viability of eggs, frequency of integration),

but it helps to avoids chimeric animals

injecting fertilized eggs
Injecting fertilized eggs
  • The eggs are harvested from mice (superovulated or natural matings).
  • The DNA is usually injected into the male pronucleus.
  • The eggs can be transferred in the same day (1 cell) or the next day (2-cells) into pseudopregnant female oviducts.
breeding t ransgenic animals transgenic founders
Breeding Transgenic animals(transgenic founders)
  • Transgenic animals Individually are backcrossed to non-transgenic animals.
  • DO NOT intercross different founders.Each founder results from a separate RANDOM transgene integration event.
slide12
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animals?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animal.
slide13

Conditional Transgenic mouse

The expression of transgene in transgenic mouse can be induced

important considerations for conditional transgenes
Important Considerations for Conditional Transgenes
  • Transgenes have low or no expression when not induced
  • Large difference between induced and non-induced gene expression
  • Transgene expression rapidly turns on or off.
  • Inducer (doxycycline, tamoxifen, cre) is not toxic and easily administered
slide15

Tetracycline Controlled Transactivator tTA

“Tet-off”

tetR

VP16

Doxycycline blocks tTA DNA binding

tTA binds to tetO to activate transcription

slide16

Reverse Tetracycline Controlled Transactivator tTA

“Tet-on”

rtetR

VP16

Doxycycline allows rtTA to bind to tetO

Without doxcycline rtTA can not bind to tetO

tetracycline regulation summary
Tetracycline Regulation: Summary

No Doxycycline Doxycycline

tTAexpressednot expressed

rtTAnot expressedexpressed

slide18
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animal.
applications of transgenic animals
Applications of Transgenic Animals

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

3 Study gene function

Many human diseases can be modeled by introducing the same mutation into the mouse. Intact animal provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing.

4. Drug testing

example 1 transgenic cattle
Example 1: Transgenic Cattle
  • Cloned transgenic cattle produce milk with higher levels of beta-caein and k-casein

Published in Nature, Jan, 2003

example 2 transgenic mouse
Example 2: Transgenic Mouse

The growth hormone gene has been engineered to be expressed

at high levels in animals.

The result: BIG ANIMALS

Mice fed with heavy metals are 2-3 times larger

Metallothionein

promoter

regulated by heavy metals

slide24

Example 3: Transgenic 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.

example 4 gfp transgenic mouse nagy
Example 4: GFP transgenic mouse (Nagy)

9.5 day embryos -

GFP and wt

Tail tip

slide27

Example 5: Wild and domestic trout respond differently

to overproduction of growth hormone.

So, GH is not effective to domestic trout.

example 6 transgenic mice as tools
Example 6: Transgenic mice as tools
  • Normal mice can't be infected with polio virus. They lack the cell-surface Polio virus receptor. But, human has Polio virus receptor.
  • Transgenic mice expressing the human gene for the Polio receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans
slide29
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animals.
knock out animal
knock-out Animal

One endogenous gene in an animal is changed. The gene can not be expressed and loses its functions.

  • DNA is introduced first into embryonic stem (ES) cells.
  • ES cells that have undergone homologous recombination are identified.
  • ES cells are injected into a 4 day old mouse embryo: a blastocyst.
  • Knockout animal is derived from the blastocyst.
slide31
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animals.
vector design
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 to knock out the gene functions.
    • Vector DNA to enable the molecules to be inserted into host DNA molecules
knockout mice
KNOCKOUT MICE

Isolate gene X

and insert it into vector.

Inactivate the gene

by inserting a marker gene

that make cell resistant

to antibiotic (e.g. Neomycin)

Normal (+) gene X

Genome

Defective (-)

Gene X

Transfer vector

with (-) gene X

into ES cells

(embryonic stem cells)

VECTOR

e.g.(NeoR)

MARKER GENE

vector and genome will recombine via homologous sequences
Vector and genome will recombine via homologous sequences

Genomic gene

Exon 4

Exon 2

Exon 3

Exon 1

Homologous recombination

and gene disrution

Grow ES cells in

antibiotic containing media;

Only cell with marker gene

(without normal target gene)

will survive

problems with homologous recombination

Solution: Replacement vectors

The knock-out construct contains the 1) NeoR gene

flanked by 2) two segments of the target gene

and 3) the HSVtk gene

Part of the gene replaced with NeoR

ES cells are selected for integration of NeoR and

against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir

Problems with homologous recombination

Unwanted random non-homologous recombination

is very frequent.

This method provides no selection against it

slide37

Homologous

recombination

Random integration

NeoR

NeoR+/ HSVtk-

NeoR+/ HSVtk+

HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells

Replacement vectors

Gene segment 1

Gene segment 2

NeoR

Linearized replacement plasmid

HSVtk

typical ko vector
Typical KO vector

*tk:thymidine kinase

inject es cells with gene x into early mouse embryo
Inject ES cells with (-) gene X into early mouse embryo

Transfer embryos

to surrogate mothers

Resulting chimaras

have some cells

with (+) gene X

and (-) gene X.

Mate them with normal mice

It is lucky,

if germline contain (-) gene X

Screen pups to find -/+ and mate them

Next generation will split as 3:1

(Mendelian)

embryonic stem cells
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 are transformed
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
implantation of blastocysts
Implantation of blastocysts
  • The blastocysts injected with transformed ES cells are left to rest for a couple of hours
  • Expanded blastocysts are transferred to the uterine horn of a pseudopregnant female
  • Max. 1/3 of transferred blastocysts will develop into healthy pups
testing the offspring
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
Breeding Chimeras (knock-out founder)

Chimera - the founder

  • germ-line transmission - usually the ES cells are derived from a 129 mouse strain (agouti or white colour) and the ES cells are injected into blastocyst derived from a C57Bl/6 mouse (black).
  • The more that the ES cells contribute to the genome of the knockout mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped.
breeding chimeras knock out founder1
Breeding Chimeras (knock-out founder)
  • Males that are 40% to 100% based on agouti coat colour should be bred
  • Females should not be bred (low incidence of success).
  • Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting knockout gene, the knockout gene will not likely go to germline and should not be used for more breeding.
littermates
Littermates

Black mouse -

no apparent ES cell

contribution

Chimeric founder -

strong ES cell

contribution

Chimeric founder -

weaker ES cell

contribution

slide52
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animal.
conditional knock out animals how to make floxed gene

Gene of interest

Conditional knock-out animals How to make FLOXed gene

TK

NeoR

Electroporate targeting vector

into ES cells, followed

by +/- selection

loxP

loxP

NeoR+/ HSVtk-

cells selected

loxP

loxP

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity.

mate floxed mice with mice carrying a cre transgene
Mate FLOXed mice with mice carrying a Cre transgene

Marker gene

Promoter elementsCre IRES GFP SV40 p(A)

intron

Crucial element. Recombinase

would be expressed in accordance

with specificity of your promoter.

Promoter could be regulated !!!

artificailly or naturally

conditional knock out animals
Conditional knock-out animals

inactivate a gene only in specific tissues

and at certain times during development and life.

Your gene of interest

is flanked by 34 bp loxPsites (floxed).

If CRE recombinase expressed

Gene between loxP sites is removed

slide56
Transgenic animals and knockout animals

Part 1: transgenic animals:

  • Introduction to transgenic animals.
  • How to make transgenic animals?
  • How to make conditional transgenic animal?
  • Applications of transgenic animals.

Part 2: Knockout animals

  • Introduction to knockout animals.
  • How to make knockout animals?
  • How to make conditional knockout animals?
  • Applications of knockout animal.
applications of knock out animals
Applications of Knock-out animals
  • Find out if the gene is indispensable (suprisingly many are not!)
  • Check the phenotypes of knockout animals
  • Determine the functions of knockout gene.
health monitoring programs
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 programs1
Health Monitoring Programs
  • Months of research data may have to be thrown out because of undetected infection:
    • Unfit for research
    • Data unreliable
pathogens
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!
pathogens some common pathogens and their effects
Pathogens: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
Pathogens (cont):Some common pathogens and their effects
  • Infected shortly after birth
  • stop breeding
  • Altered physiology: as the virus travels down the respiratory tract -necrosis of airway epithelium, pneumonia in lungs, lesions.
  • 129/J and DBA, aged and immunodeficient mice most susceptible; SJL/J and C57Bl/6 most resistant
pathogens cont some common pathogens and their effects1
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
    • Wound healing
pathogens cont some common pathogens and their effects2
Pathogens (cont):Some common pathogens and their effects
  • MHV
    • Probably most important pathogen of laboratory mice
    • Extremely contagious; aerosol, direct contact;
    • No carrier state
    • Clinic state: varies dependent upon MHV and mouse strains
pathogens cont some common pathogens and their effects3
Pathogens (cont.):Some common pathogens and their effects
  • Diarrhea, poor growth, death
  • Immunodeficient (e.g. nu/nu) wasting syndrome -eventual death
  • 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, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc.
pathogens cont some common pathogens and their effects4
Pathogens (cont.):Some common pathogens and their effects
  • Helicobacter spp
    • H. Hepaticus (mice) most prominent
    • Transmission: direct fecal-oral
    • Clinical signs absent in immunocompetent mice
    • In immunodeficient mice- rectal prolapse
    • Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms
pathogens cont some common pathogens and their effects5
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 effects6
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 effects7
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