BIOTECHNOLOGY -intentional manipulation of genetic material of an organism.
Genetic Manipulation - University of Western Australia...•Manipulation of gene expression by...
Transcript of Genetic Manipulation - University of Western Australia...•Manipulation of gene expression by...
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Genetic Manipulation
Anke van Eekelen, PhD
Telethon Institute for Child Health Research
100 Roberts RdSubiaco, WA 6008
9489 7886, [email protected]
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WHY
would you make a genetically manipulated animal ?* To study the gene identity - gene function relationship
•Manipulation of gene expression by genetic manipulation creates animal models (in vivo models), which reveal a transgenic phenotype of the animal
•This transgenic phenotype is the combination of a set of observed characteristics of the animal resulting from transgenesis: → biochemistry
→ anatomy → physiology → behaviour
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Gain of Function-model : Additional copy of a gene - overexpressionAberrant form of a gene - targeted gene mutation
Loss of Function-model : Gene deletion by replacement - knockout animal
These models may reveal the mechanism/pathwayunderlying
a specific outcome or disease
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HOW
to make a genetically manipulated animal?
Transgenic mice: pronuclear/oocyte injectionof targeting vector/construct containing the DNA of interest
Knockout mice : blastocyst injectionof transformed embryonic stem cells expressing your gene of interest
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Pronuclear injection to make transgenic mouse
Foreign DNA injected is a construct/vector containing:- full coding sequence of the gene of interest- promotor determining tissue specificity & strength
of expression
* Site of integration of injected DNA into genome is random!
* Number of copies of injected DNA into genome is random!single insertion - tandem (>100) array
range of foreign gene expressionrange of phenotypes
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• developed transgenics = founders
• Founders are hemizygous fortransgene!
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2 cell stage 8 cell stageMORULA
16 cell stageMORULA
BLASTULAIntegration of transgene before first cell division
↓developing embryo will have transgene present inevery somatic & germ line cell(10-30%) Integration of transgene after 2 cell stage
↓Developing embryo = chimera (genetic mosaic)(10%)
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Cloning(Ultimate transgenesis)
transfer of a complete “somatic cell nucleus”transfer of a complete “somatic cell nucleus”
www.ozgene.com
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blastocystblastocyst
complete adult udder cell fused with an enucleated oocyte by electrical
pulseOocyteOocyte
Sheep “Dolly”Sheep “Dolly”somatic cellsomatic cell
Wilmut, I., et al.,’98
www.ozgene.com
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HOW
to make a genetically manipulated animal?
Transgenic mice: pronuclear/oocyte injectionof targeting vector/construct containing the DNA of interest
Knockout mice : blastocyst injectionof transformed embryonic stem cells expressing your gene of interest
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Transgenic mouse versus Knockout mouse
- random integration into genome
-Tandem arrays= multiple copies
- fertilized oocyte- site specific integration of transgene
- homologous recombination
replacement vector containing:* two flanking regions of DNA homologous
to the genomic target locus* positive and negative selection markers
- blastocyst
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Blastocyst
Inner Cell Mass (ICM) develops into embryo
blastocoel
trophectoderm
www.ozgene.com
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Gene-knockout/in procedure in a nutshell
www.ozgene.com
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Bacterial gene neo (neomycin phosphotransferase)Causes resistence to drug G418
Tyrosine kinase gene causes sensitivity to drug gancyclovir
Homologous Recombination
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1st generation = F1 = founder generation
Blastula injection to makeknockout mousePluripotent murine embryonic
stem cells from blastocyst→
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Reporter Mice ?
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Okabe M, Okabe M, IkawaIkawa M et al ‘97M et al ‘97
www.ozgene.com
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Transgenic reporter mice-A: randomly integrated reporter geneunder ubiquiteous promoter(green mouse phenotype)
-B: randomly integrated reporter geneunder cell specific promoter (spatial control)
Knockin reporter mice: * Knockout and knockin at same time* Replacement construct contains:
* two flanking regions of DNA homologous to the genomic target locus* positive and negative selection markersBut also….* full coding sequence of another gene than target gene
→ this new gene will be under control of the promotor of the target gene
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Knockin reporter mouse
Scl allele:
LacZ KI
1a 1b 2 3 4 5 6 exon
Whole mount LacZ staining LacZ staining on cryosections
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Gene deletion in knockout model → mouse phenotype
PhenotypicdifferenceLethal Normal
Targeted gene is eitherunimportant
or redundant
Targeted gene is critical for
development/ survival
Function of targeted gene is revealed
Conditional gene deletion
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Conditional gene deletion model
Alternative approach to conventional knockout gene deletion is required:
↓Spatial and/or temporal control over gene deletion
Condition gene deletion models:
* mice express a combination of transgenic and knockin alleles* simultaneous expression of these genetically manipulated alleles
underlying gene deletion*gene deletion is happening in vivo !!
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Spatial and/or temporal control over gene deletion
* tissue specific promotorcontrols transgene expression
* developmental stage specific promotorcontrols transgene expression
* inducible activity of expressed transgene
Different conditional gene deletion models: * Cre-loxP / CreER(T)* Tet-On or Tet-Off
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Cre-loxP system
Principle: gene deletion by DNA recombination
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Cre-loxP system
Cre recombinase LoxP - DNAsequence
* Bacteriophage P1 * Two 13 bp inverted repeats interupted by 8 bp
* mediates site specific nonpalindromic sequence recombination between (34 bp in total length)two lox P sites
TRANSGENE KNOCKIN
ATA ACT TCG TAT AATA ACT TCG TAT A gcgc at ac atat ac at T ATA CGA AGT TATT ATA CGA AGT TAT
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Cre-LoxP DNA recombination
crecre++loxP loxP
++loxP
loxP
www.ozgene.com
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Knockin mouse= floxed mouse
Transgenic mouse= Cre-mouse
Gene X floxed / Cre+
Genomic sequence for gene X is recombined: Gene X expression
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Example: brain specific KO of SCL gene(Cre-loxP; spatial)
Nestin promotor
Conditional SCL-KO in brain
Nestin: * pro-neural gene* specific expression
in brain tissue
Scl: * hematopoietic regulator* expressed in brain tissue* knockout is lethal
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Inducible transgenic mouse modelCreER(T)-loxP system
(spatial & temporal)
Tissue specific promoter + Cre ER(T)* Spatial * Temporal
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ER(T): mutated estrogen binding domain with affinity only for Tamoxifen (= estrogen antagonist) ↓
Cre ER(T) : * fusion protein behaving like a steroid receptor
* Tamoxifen binding to Cre ER(T) in cytoplasminduces translocation of of cre to nucleus
* In cell nucleus cre can achieve DNA recombination of a floxed gene
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Cre ER(T)
Cre ER(T) TAM
Cre-ERTTissue spec. propmoter
lox P sites
Gene X: allele 1
TAM
Inducible Cre-ER(T) / loxP model for gene deletion
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Inducible gene deletion models
* Cre-loxP based models
Tamoxifen CreER(T)
Alternative inducers for Cre recombinase:
RU486 CrePRRU486 or Dexamethasone CreGRInterferon Mx1promoter-Cre
* Tet-based models
Tetracycline Tet-On or Tet-Off
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In summary:
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Transgenic mouse: random genomic integration of transgene
Knockout/in mouse: site-specific genomic integration of transgene(targeted)
Conditional transgenic: tissue or time specific expression of transgene(random or targeted)
Inducible transgenic: tissue and time specific expression of transgene(random and targeted)
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Alternative gene manipulation approaches:
1- RNAi
2- Morpholinos/antisense hybridisation
Alternative approach to make transgenic mice:
1- lentiviral infection (retrovirus, which incorporates in genome)
post transcriptional gene silencing
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RNA interference pathway
siRNA
‘Dicer’ ribonuclease
RNA-induced silencing complex
(RISC)
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Morpholino Antisense oligos
- bind and inactivate selected RNAs
- fast, simple and most effective for validation of new therapeutic targets(modern drug development)
- potentially effective therapeutics inviral diseases and cancer
-Specific, stable and no non-antisense activity* Morpholino ring with 1 of 4 genetic bases
(replacing the ribose backbone of endogenous RNA)* non-ionic phosphorodiamidate inter-subunit
But … NO GERMLINE TRANSMISSION!!!
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Lentiviral genetic manipulation
““TransgenesTransgenes go retro” Nature Biotech Jan. ‘99go retro” Nature Biotech Jan. ‘99
www.ozgene.com
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Lentiviraltransgenesisof siRNA
RNA interference pathway
siRNA
‘Dicer’ ribonuclease
RNA-induced silencing complex
(RISC)