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Lecture 1
Overview of early mouse development and methodology
nb reading list is at end of notes for this lecture
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Vertebrate development – classical models
1mm
1 cm 100 microns
Phylotypicstage
Similar Similar
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Development = origami using layers/sheets of cells
Three germ layers, Ectoderm, Mesoderm, and Endoderm (derm=layer), give rise to all cells and tissues of the developing embryo.
Vertebrates are triploblasts
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Why study the mouse?
• The Victorian mouse fancy movement provided a ready made resourceof inbred strains, variants and mutants
• Fast generation time (21 day gestation)
• Amenable to genetic manipulation
• Tissue culture models
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Positional informationCell Fate
Anterior (Head)
Posterior (Tail)
Dorsal (Back)Ventral (Front)
Left
Right
Identity and location
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Mammals have extensive extraembryonic tissues
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In utero development in mouse occurs over 19-21 days
• E (embryo stage) = dpc (days post coitum). Most commonly referred to from 0.5 onwardsas mating takes place at night.
• Preimplantation development occurs up to E3.5. All other development occurs postimplantation.
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Preimplantation Development
Trophectoderm
Primitive (primary) endoderm
Inner cell mass/Primitive ectoderm
Cleavage stages
Zona pelucida
Blastocoel cavity
Activation of embryonic genome
Blastomere
0 1 2 3 4 days
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Early Post-implantation Development
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Gastrulation and Beyond
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Confusing nomenclature!
A ‘derm’ is a cell layer – not a cell type!
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Our understanding of the world can only be as good as the state of the art technology we use to measure it – knowledge is relative, not absolute.
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Experimental Tools for studying mouse embryos
Embryological approaches; • Histological analysis and conventional microscopy
• Cell fate mapping (dyes and now tagged loci)
• In vitro culture of preimplantation stages and in some cases postimplantation stages.
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• Chimera formation and embryo aggregation.
• Cell culture models
e.g. tetraploid chimeras for testing gene function in extraembryonic vs embryonic lineages.
Embryological approaches;
Embryonic stem (ES) cells
• Embryo manipulation/transplantation
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• In situ hybridization
• Immunohistochemistry
Eed + NanogOct4 + Eed
Sections Wholemount
Molecular embryology;
• Gene expression profiling of embryos, dissected fragments, derivative tissue culture cell lines and single cells.
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Genetic approaches;• Classical mouse mutants
Brachyury mouse with short tail is dominant mutation in gene fortranscription factor required for mesoderm formation.
• Genetic screens
Wild-type and Nodal (d/d) mutant embryos with staining for markers of primitive streak (brown) and ectoderm (dark blue).
Chemical (ENU) mutagenesis – requires lengthy genetic mapping and cloning to identify mutated locus
Insertional or ‘gene trap’ mutagenesis in ES cells – can go directly to gene of interest
SA
SD
Antibiotic resistancemarker
Reporter gene
IRESPolyA signal
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• Production of transgenic mice by pronuclear injection of DNA
- Gene construct injected into male pronucleus of 1-cell embryo
- DNA integrates randomly at single site, usually multicopy
Genetic manipulation in mouse;
• Production of genetically modified mice by transferring ES cells to recipient embryo
- Gene manipulation using homologous recombination in ES cells
- Inject modified cells intoRecipient embryo to produce chimeric animal that transmits donor genome through the germ-line.
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• Gene targeting in embryonic stem (ES) cells
Genetic manipulation in mouse;
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Conventional gene knockout strategy (replacement vector)
X X
Positive selectableMarker gene
Negative selectableMarker gene
Knock-out (or Knock-in)
Genetic manipulation in mouse;
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Positive selectableMarker gene
Negative selectableMarker gene
X X
+ site specific recombinase (Cre or Flp)
+
Recombinase recognition sequence
Conditional gene knockout strategy;
Genetic manipulation in mouse;
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Homozygous conditional allele Transgenic mouse expressing site specific recombinasein tissue specific pattern
X
Analyse phenotype in F1 embryos or adults
Examples of recombinase driver transgenics;
- Cre recombinase driven by Nanog promoter
- Estrogen receptor-Cre recombinase fusion driven by constitutive promoter. Addition of Tamoxifen to drinking water triggers nucleartranslocation of recombinase giving temporal control of gene deletion.
Conditional gene knockout strategy;
Genetic manipulation in mouse;
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Textbook; Principles of Development, Lewis Wolpert and Cheryl Tickle. Review papers;Lecture 1 -3 Alexandre (2001) International Journal of Developmental Biology 45, p457-467 Rossant (2001) Stem Cells 19, p477-82 Yamanaka et al, (2006). Developmental Dynamics 235, p2301-2314 Katsuyoshi and Hamada, (2012) Development 139, p3-14 Lecture 4 and 5 Arnold and Robertson (2009) Nature reviews Molecular cellular biology, 10, p91-103 Robb and Tam (2004) Seminars in Cell and Developmental biology 15, p43-54 Hayashi et al (2007) Science 316, p394-396. Hashimoto and Hamada (2010) , Curr Opin Genet Dev 20, p433-7
Hanna et al (2010) Cell 143, p508-525. Yamanaka and Blau (2010) Nature 465, p704-712
Reading list
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New innovations in ES cell manipulation(optional if time permits)
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ZFN, TALEN and CrispR/cas systems;
Genetic manipulation in mouse;
• Cys2-His2 zinc finger domain contacts 3bp of sequence in major groove with varying levels of selectivity.
• Can use as modular component to get sequence specific targeting of Fokl restriction endonuclease monomer. Cleavage requires targeting second monomer to other strand to generate functional Fokl dimer.
• Provides substrate for error prone repair or HR
using recombinant DNA template for custom modification.
• TALE effector proteins secreted by Xanthomonas bacteria in order to activate host plant gene expression that aids infection.
• Modular composition of sequence specific binding domains comprising 33-34 amino acids with positions 12 and 13 being highly variable.
• Can be used to construct designer Transcription Activator Like Effector Nuclease (TALEN) to introduce DNA breaks at defined target sequence.
• Provides substrate for error prone repair or HR using recombinant DNA template for custom modification.
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• RNA mediated bacterial defense against viral or plasmid DNA.
• Type II system adapted for genome engineering in many organisms.
• Can use cas9 intrinsic nuclease to introduce ds break or ss nick.
• Provides substrate for error prone repair or HR using recombinant DNA template for custom modification.
• Can also mutate directly by injection into zygote.
• Partially circumvents requirement for highly recombinogenic cell such as ES cell.
ZFN, TALEN and CrispR/cas systems;
Genetic manipulation in mouse;
PAM site
(Trans-encoded CRISPR RNA)