Manipulation of gene expression by genetic manipulation creates animal models
(in vivo models), .... Principle: gene deletion by DNA recombination ...
Genetic Manipulation of Mice Anke van Eekelen, PhD Telethon Institute for Child Health Research
100 Roberts Rd Subiaco, 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 - overexpression Aberrant form of a gene - targeted gene mutation Loss of Function-model : Gene deletion by replacement - knockout animal
These models may reveal the mechanism/pathway underlying a specific outcome or disease
HOW
?
to make a genetically manipulated animal
Transgenic mice: pronuclear/oocyte injection of targeting vector/construct containing the DNA of interest Knockout mice : blastocyst injection of 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 expression range of phenotypes * developed transgenics = founders * Founders are hemizygous for transgene!
2 cell stage
8 cell stage MORULA
16 cell stage MORULA
BLASTULA
Integration of transgene before first cell division
↓ developing embryo will have transgene present in every somatic & germ line cell (10-30%)
Integration of transgene after 2 cell stage
↓ Developing embryo = chimera (genetic mosaic) (10%)
HOW
?
to make a genetically manipulated animal
Transgenic mice: pronuclear/oocyte injection of targeting vector/construct containing the DNA of interest Knockout mice : blastocyst injection of transformed embryonic stem cells expressing your gene of interest
Transgenic mouse versus Knockout mouse
- random integration into genome - Tandem arrays= multiple copies
- 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 ($$)
($$$$$$)
Gene-knockout/in procedure in a nutshell
Homologous Recombination
Bacterial gene neo (neomycin phosphotransferase) Causes resistence to drug G418 Tyrosine kinase gene causes sensitivity to drug gancyclovir
Pluripotent murine embryonic stem cells from blastocyst →
Blastula injection to make knockout mouse
1st generation = F1 = founder generation
Increasingly more control over reporter gene expression
Reporter Mice (LacZ, GFP) Transgenic reporter mice-A: randomly integrated reporter gene under ubiquiteous promoter (green mouse phenotype) -B: randomly integrated reporter gene under 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 markers But also…. * full coding sequence of another gene than target gene → this new gene will be under control of the promotor of the target gene
Example: Knockin reporter mouse LacZ KI
SCL allele: 1a 1b 2
Whole mount LacZ staining
3
4
5
6
exon
LacZ staining on cryosections
Gene deletion in knockout model → mouse phenotype
Lethal
Targeted gene is critical for development/ survival
Phenotypic difference
Function of targeted gene is revealed
Conditional gene deletion
Normal
Targeted gene is either unimportant or redundant
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 promotor controls transgene expression *
developmental stage specific promotor controls 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 nonpalindromic sequence (34 bp in total length)
* mediates site specific recombination between two lox P sites
TRANSGENE
KNOCKIN
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
SCL: * hematopoietic regulator * expressed in brain tissue * knockout is lethal
Nestin: * pro-neural gene * specific expression in brain tissue
Inducible transgenic mouse model CreER(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 cytoplasm induces translocation of of cre to nucleus * In cell nucleus cre can achieve DNA recombination of a floxed gene
Inducible Cre-ER(T) / loxP model for gene deletion Cre
Tissue spec. propmoter
ER(T)
TAM
Cre-ERT
Cre
ER(T)
Gene X: allele 1
lox P sites
TAM
Inducible gene deletion models * Cre-loxP based models Tamoxifen
CreER(T)
Alternative inducers for Cre recombinase: RU486 RU486 or Dexamethasone Interferon
CrePR CreGR Mx1promoter-Cre
* Tet-based models Tetracycline
Tet-On or Tet-Off
increased control over gene deletion
In summary: 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: post transcriptional gene silencing 1- RNAi 2- Morpholinos/antisense hybridisation
Alternative approach to make transgenic mice: 1- lentiviral infection (retrovirus, which incorporates in genome)
RNA interference approach for posttranscriptional gene silencing
Step 1: dsRNA cleavage by Dicer
Step 2: recruitment of siRNA & RNAi factors and formation of RISC Step 3: siRNA-unwinding and RISC activation
Step 4: mRNA targeting and degradation
RISC: RNA-induced silencing complex
Morpholino Antisense oligomers -
contain a 6 member morpholine ring (non-ionic phosphorodiamidate inter-subunit) instead of a 5 member ribose or deoxyribose sugar in RNA and DNA
- this structure gives resistence to nucleases, thus stay intact and function longer! - stringent hybridisation with mRNA independent of salt concentrations
-
designed as antisense RNA oligos to bind and inactivate selected mRNAs
- objective is to inhibit mRNA translation But injection of morpholino is only transient KO … NO GERMLINE TRANSMISSION!!!
Literature: What’s wrong with my mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. Jaqueline N. Crawley Wiley-Liss, 2000
4
Manipulating the Mouse Embryo. A Laboratory Manual (2nd Edition). Brigid Hogan, Rosa Beddington, Frank Constantini & Elizabeth Lacy Cold Spring Harbor Laboratory Press, 1994
4
