Genetic Manipulation
Anke van Eekelen, PhD
Telethon Institute for Child Health Research
100 Roberts RdSubiaco, WA 6008
9489 7886, [email protected]
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
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
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
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
• developed transgenics = founders
• Founders are hemizygous fortransgene!
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%)
Cloning(Ultimate transgenesis)
transfer of a complete “somatic cell nucleus”transfer of a complete “somatic cell nucleus”
www.ozgene.com
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
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
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
Blastocyst
Inner Cell Mass (ICM) develops into embryo
blastocoel
trophectoderm
www.ozgene.com
Gene-knockout/in procedure in a nutshell
www.ozgene.com
Bacterial gene neo (neomycin phosphotransferase)Causes resistence to drug G418
Tyrosine kinase gene causes sensitivity to drug gancyclovir
Homologous Recombination
1st generation = F1 = founder generation
Blastula injection to makeknockout mousePluripotent murine embryonic
stem cells from blastocyst→
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
Incr
easi
ngly
mor
e co
ntro
l ove
r rep
orte
r gen
e ex
pres
sion
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
Knockin reporter mouse
Scl allele:
LacZ KI
1a 1b 2 3 4 5 6 exon
Whole mount LacZ staining LacZ staining on cryosections
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
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 !!
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
Cre-loxP system
Principle: gene deletion by DNA recombination
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
Cre-LoxP DNA recombination
crecre++loxP loxP
++loxP
loxP
www.ozgene.com
Knockin mouse= floxed mouse
Transgenic mouse= Cre-mouse
Gene X floxed / Cre+
Genomic sequence for gene X is recombined: Gene X expression
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
Inducible transgenic mouse modelCreER(T)-loxP system
(spatial & temporal)
Tissue specific promoter + Cre ER(T)* Spatial * Temporal
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
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
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
In summary:
i nc r
e as e
d co
ntr o
l ove
r gen
e de
l eti o
n
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)
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
RNA interference pathway
siRNA
‘Dicer’ ribonuclease
RNA-induced silencing complex
(RISC)
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!!!
Lentiviral genetic manipulation
““TransgenesTransgenes go retro” Nature Biotech Jan. ‘99go retro” Nature Biotech Jan. ‘99
www.ozgene.com
Lentiviraltransgenesisof siRNA
RNA interference pathway
siRNA
‘Dicer’ ribonuclease
RNA-induced silencing complex
(RISC)
Top Related