transgenic animals and knockout animals. 3 main ways to do biological research: 1.do research in...

Transgenic animals and knockout animals

3 main ways to do biological research:

1. Do research in test tubes.

2. Do research with cells.

3. Do research directly with 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

• Animal has one or more foreign genes inserted into chromosome DNA inside its cells artificially.

• 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


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.

ES cell transformation

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

3. Injection of gene into fertilized egg is less reliable (viability of eggs, frequency of integration),

but it helps to avoids chimeric animals

2. ES cell transformation provides more control of the integration step (selection of stably transfected ES cells)

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 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.


Part 1: transgenic animals:• Introduction to transgenic animals.• How to make transgenic animals?• How to make conditional transgenic animals?• Applications of transgenic animals.


Conditional Transgenic mouse

The expression of transgene in transgenic mouse can be induced

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

Tetracycline Controlled Transactivator tTA

“Tet-off”

Doxycycline blocks tTA DNA binding

tTA binds to tetO to activate transcription

tetR VP16

Reverse Tetracycline Controlled Transactivator tTA

“Tet-on”

Doxycycline allows rtTA to bind to tetO

Without doxcycline rtTA can not bind to tetO

VP16rtetR

Tetracycline Regulation: Summary

No Doxycycline Doxycycline

tTA expressed not expressed

rtTA not expressed expressed

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

• Cloned transgenic cattle produce milk with higher levels of beta-caein and k-casein

Published in Nature, Jan, 2003

Example 2: Transgenic Mouse

The growth hormone gene has been engineered to be expressed at high levels in animals.

The result: BIG ANIMALS

Metallothionein promoter

regulated by heavy metals

Mice fed with heavy metals are 2-3 times larger

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 3: Transgenic Mouse

Tail tip9.5 day embryos - GFP and wt

Example 4: GFP transgenic mouse (Nagy)

GFP transgenic mouse (Nagy)

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

• 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

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.

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

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)

Transfer vector with (-) gene X into ES cells

(embryonic stem cells)MARKER GENE

VECTOR

Genome

Normal (+) gene X

Defective (-)

Gene X

e.g.(NeoR)

Vector and genome

will recombine via homologous

sequences

Grow ES cells in antibiotic containing media; Only cell with marker gene

(without normal target gene) will survive

Genomic geneExon 1Exon 2Exon 3Exon 4

Homologous recombinationand gene disrution

Problems with homologous recombination

Unwanted random non-homologous recombination is very frequent.

This method provides no selection against it

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

NeoR

HSVtkGen

e seg

men

t 1

Gen

e seg

men

t 2

Linearized replacement plasmid

NeoR

Homologousrecombination

NeoR+/ HSVtk-

Random integration

NeoR+/ HSVtk+

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

Replacement vectors

Typical KO vector

*tk:thymidine kinase

Inject ES cells with (-) gene X

into early mouse embryo

Resulting chimaras have some cells with (+) gene X and (-) gene X.

Transfer embryos to surrogate mothers

Mate them with normal mice

Screen pups to find -/+ and mate them

It is lucky, if germline contain (-) gene X

Next generation will split as 3:1 (Mendelian)

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 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

Implanting blastocysts

1 2

Implanting blastocysts

3 4

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 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 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

Black mouse - no apparent ES cell contribution

Chimeric founder - strong ES cellcontribution

Chimeric founder - weaker ES cellcontribution

Chimeric mouse

Conditional knock-out animals How to make FLOXed gene

loxP loxP

NeoR TK

Gene of interest

Electroporate targeting vector into ES cells, followed by +/- selection

NeoR+/ HSVtk-cells selected

Gene flanked by loxP sites (floxed)

Make mice and breed floxed allele to homozygousity.

loxP loxP

Mate FLOXed mice with mice carrying a Cre transgene

Promoter elements Cre IRES GFP SV40 p(A) intron

Marker gene

Crucial element. Recombinase would be expressed in accordance with specificity of your promoter.

Promoter could be regulated !!!artificailly or naturally

Conditional knock-out animals

inactivate a gene only in specific tissues and at certain times during development and life.

Cre-lox technology

Cre – a site-specific recombinase enzyme from the P1 phage.

Recognises a 34bp DNA sequence loxP =

Cre

Cre

Cre

Your gene of interest is flanked by 34 bp loxP sites (floxed).

If CRE recombinase expressed

Gene between loxP sites is removed

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

• 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!

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– 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


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


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 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 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


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


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


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!

transgenic animals and knockout animals. 3 main ways to do biological research: 1.do research in...

Documents

conditional transgenic

nontransgenic animals

conditional knockout

knockout animals introduction

chimeric animals

conditional transgenic

transgenic animal animal

induced slide