HTML Injection Attacks: Impact and Mitigation Strategies
Biopol seminar v4
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. 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. Overview
• The mouse model
• Protein of interest
• Cerebellum region of the brain
• Structure
• Development
• Rationale
• Preliminary results
• Summary
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. 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. 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. 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
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. 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. 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. • 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. 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. 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. 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
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. • Significant changes in
morphology
• Isl1 misexpression
• Mechanism?
• Possible interactions with other
TFs
Summary