Development of Diagnostic and
Therapeutic Procedures
Program 5
This programme focuses on the
identification of the molecul...
Genes, Mouse Models, Brain
Development and Pathologies
Nicole Dodd
13th June 2013
Laboratory of Molecular Pathogenetics
In...
Overview
• The mouse model
• Protein of interest
• Cerebellum region of the brain
• Structure
• Development
• Rationale
• ...
The mouse model
• Relatively easy to maintain, with the ability to
multiply quickly
• The mouse is closely related to huma...
Genetic manipulation
• Gene knock out
Sequenced but unknown
function
• Cre-lox recombination
Allows genes to be
activated,...
Cre recombinase activity indicated by the ROSA26-stop-lacZ reporter
Floxed Neurod1/Atoh1
CreIsl1 Isl1
LacZ
Floxed
STOP β-G...
Pax2-Isl1 Transgene
Pax2 8.5-kb regulatory
sequence
Pax2 promoter Isl1
SalI, AatlI, NotI
pGEM7f
SalI, NotI, AatlI
8.5Size ...
Transgenics - pronuclear injection of Pax2-isl1 construct
Pax2 promoter
LIM LIM
Homeodomain
DNA bindingProtein binding
Isl...
• Transcription factor Islet1 (Isl1)
• Protein that binds to DNA to control the flow of
transcription
• Important for deve...
The Cerebellum
• Represents 10% of
total brain vol.
• Contains more than
half of our neurons
• Functions to fine
tune move...
Cerebellum location
• Located at the back of the brain, underlying the cerebral
cortex
• Develops from the dorsal region o...
• Size and structure are determined by correct number of
neurons (proliferation and apoptosis)
• Correct location of neuro...
Defects in cerebellar neuronal proliferation and
migration lead to tumour and ataxia
Medulloblastoma
• Malignant
medullobl...
Rationale
• Unravel the role of Isl1 in neurosensory development
• Due to early KO lethality, role in motor neuron develop...
0
2
4
6
8
10
12
wt T1 T2
Numberofsurviving
pups/litter
Number of surving pups
P < 0.001
• Significant difference in number...
Changes in Cerebellum
T2wt
P0
P3
Immunohistochemistry
• By staining sections with fluorescently labelled antibodies we
can identify the cells that are expr...
• Significant changes in
morphology
• Isl1 misexpression
• Mechanism?
• Possible interactions with other
TFs
Summary
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Biopol seminar v4

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Biopol seminar v4

  1. 1. Development of Diagnostic and Therapeutic Procedures Program 5 This programme focuses on the identification of the molecular causes of diseases and on the development of new procedures for the prevention and diagnostics of the diseases.
  2. 2. Genes, Mouse Models, Brain Development and Pathologies Nicole Dodd 13th June 2013 Laboratory of Molecular Pathogenetics Institute of Biotechnology AS CR v.v.i.
  3. 3. Overview • The mouse model • Protein of interest • Cerebellum region of the brain • Structure • Development • Rationale • Preliminary results • Summary
  4. 4. The mouse model • Relatively easy to maintain, with the ability to multiply quickly • The mouse is closely related to humans • Genome is organised similarly • Most human genes have functional mouse counterparts • Mutations that cause diseases in humans often cause similar diseases in mice • Gene transfer technology is highly advanced
  5. 5. Genetic manipulation • Gene knock out Sequenced but unknown function • Cre-lox recombination Allows genes to be activated, repressed, or exchanged • Transgenic Introducing an exogenous gene
  6. 6. Cre recombinase activity indicated by the ROSA26-stop-lacZ reporter Floxed Neurod1/Atoh1 CreIsl1 Isl1 LacZ Floxed STOP β-Galactosidase catalyzes the hydrolysis of X-Gal producing a blue precipitate LoxP R26R Promoter Transgenic cre-lox recombination system
  7. 7. Pax2-Isl1 Transgene Pax2 8.5-kb regulatory sequence Pax2 promoter Isl1 SalI, AatlI, NotI pGEM7f SalI, NotI, AatlI 8.5Size (kb): 1.6 0.8 3.4 lacZ mRNA tag NotI Wnt1 poly A • Islet1 knock-out is lethal in early development • Generated transgenic mice
  8. 8. Transgenics - pronuclear injection of Pax2-isl1 construct Pax2 promoter LIM LIM Homeodomain DNA bindingProtein binding Isl1 FVB T2 T1 -/+ -/+
  9. 9. • Transcription factor Islet1 (Isl1) • Protein that binds to DNA to control the flow of transcription • Important for development of a number of organs • Expressed in ganglion neurons • Required for various aspects of motor neuron development • Represses genes involved in neurogenesis Protein of interest
  10. 10. The Cerebellum • Represents 10% of total brain vol. • Contains more than half of our neurons • Functions to fine tune movement and balance • Plays a major role in the coordination of muscle activity
  11. 11. Cerebellum location • Located at the back of the brain, underlying the cerebral cortex • Develops from the dorsal region of the posterior neural tube • Cells arise from two different germinal matrices
  12. 12. • Size and structure are determined by correct number of neurons (proliferation and apoptosis) • Correct location of neurons (migration) • Cell type (differentiation) Brain (cerebellum) development Bergman glia Purkinje layer External granular layer Molecular layer Internal granular layer
  13. 13. Defects in cerebellar neuronal proliferation and migration lead to tumour and ataxia Medulloblastoma • Malignant medulloblastomas are the most common malignant brain tumour of childhood • Thought to originate from granule neuron precursor cells the developing cerebellum
  14. 14. Rationale • Unravel the role of Isl1 in neurosensory development • Due to early KO lethality, role in motor neuron development is unclear • Determine the effects of ectopic expression in the cerebellum on proliferation, migration & differentiation • Defects in cerebellar neuronal proliferation and migration can lead to cancer
  15. 15. 0 2 4 6 8 10 12 wt T1 T2 Numberofsurviving pups/litter Number of surving pups P < 0.001 • Significant difference in number of surviving pups • Transgenic expression of Isl1 affects embryonic development and postnatal survival of transgenic pups Number of surviving pups
  16. 16. Changes in Cerebellum T2wt P0 P3
  17. 17. Immunohistochemistry • By staining sections with fluorescently labelled antibodies we can identify the cells that are expressing the transgene T1 P3 mt T1 P3 wt
  18. 18. • Significant changes in morphology • Isl1 misexpression • Mechanism? • Possible interactions with other TFs Summary

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