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Transcript
Neurobiology of
Learning and Memory
Prof. Stephan Anagnostaras
Lecture 9: Molecular-Genetic
Approaches to Learning & Memory
CamKIIa and CREB
are required for LTP
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Mechanisms of Learning and Memory
Synaptic
Kinase
activation
A
NMDA
D1
AMPA
Cyclase
cAMP
CaMKII
PDE
Nuclear CREB
activation
Ca
CREB
Gene replacement and transgenic
animals
• Some genes are identified through mutant analysis
Forward Genetics (mutant phenotype---> genotype)
• To determine the function of these genes, it is possible
to replace an organism’s wild type gene with an inactive
gene to create a “gene knockout”
Reverse Genetics (mutant genotype--->phenotype)
• It is also possible to introduce additional genes
(transgenes) to create a transgenic organism
• Epigenetics
Associate gene expression or polymorphisms with phenotype
In mice, two main kinds of animals
Transgenic
A novel gene, the transgene, either man-made or
borrowed from another animal is added to the genome.
Works in many species - easy
Knockout
A gene is deleted; in practice the gene is replaced in the
same location (homologous recombination) with a null
mutation of the gene. Only possible in mice, difficult
Related mutants:
Dominant negative: a massively overexpressed transgene
that interferes with the endogenous gene (easy)
Knockin: Homologous recomination of a transgene (very
difficult and rare)
In vitro mutagenesis of a cloned gene
Gene knockout and
transgenic techniques
usually involve
mutagenesis
of cloned genes prior to
transfer into the
organism
Production of
transgenic mice
Confusing example
is a dominant
negative transgene
(like Mayford &
Kandel), not a
knockout but a lot
like a knockout
Creation of mice embryonic stem (ES) cells
carrying a knockout mutation
Gene knockout in
mice
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Qu ic kTime ™ an d a TIFF (U n co mpre ss ed ) d ec o mpre ss or are n ee de d to s ee this picture .
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T=Threonine (ACT, ACC, ACA)
A=Alanine (GCT, GCC, GCA,GCG)
Disruption of CamKIIa
by T286A point
mutation disrupts LTP
and spatial memory
•Disrupts
autophosphorylation
of Threonine at 286
Unable to switch to
Ca-Calmodulin independent state
Giese et al., Science,1998
Cell-type-specific
gene knockouts in
mice: Cre-Lox
technique
Spatial and/or
temporal control
of deletion
Tsien et al., Cell, 1996: CA1 specific
NMDAR1 knockout
CA1
Tsien et al.,
Cell, 1996
DG
McHugh et al., Cell, 1996:
Place cells in CA1 NMDAR KO
Abnormally large place fields
Low coherent firing
An Temporally controlled inducible CREB
Repressor: The LBD Fusion Approach
Kida, Josselyn
et al., 2002
LBD
Heat Shock
Protein
CREBr
Tamoxifen
LBD
Cytoplasm
CREBr
LBD
CREBr
Nucleus
The LBD-CREB-MT Vector
(single Transgene)
 CaMKII promoter
HA
LBD
CREB-MT
intron
polyA signal
HA; influenza virus hemagglutinin (HA) tag
LBD ; mutant estrogen receptor ligand binding domain (G521R)
CREB-MT; dominant negative CREB (CREB S133A)
Josselyn et al., 2002 - temporal control
of the CREB repressor
Fear conditioning
Tetracycline-regulated
expression of CamKIIaAsp286 (dominant
negative, always
phosphorylated T286D
D= Aspartate,
“constitutively active”)
Double-transgenic
• Cell-specific tTA
• tetO + transgene to regulate
Mayford & Kandel, TIG, 1999
D= GAT, GAC
Tet-Off System
Summary - Reverse Genetics
•Molecular Biology Approaches
Mutant Mice
• Knockouts - global deletion
• Transgenics - global addition
Later generation:
• Point mutants (PointLox method)
• Region-specific deletion by Cre-Lox
• Inducible deletions
• Temporal and or region control of transgene
- LBD fusion approach
- Tet system
Other Approaches - Quantitative
Genetics and Functional Genomics
Forward Genetic Approaches
QTL (Quantitative Trait Loci)
ENU (ethylnitrosurea - random mutagenesis)
Dominant & recessive screens
Mapping & Positional Cloning
Epigenetic approaches
DNA Microarrays
Current mouse array = 11k genes/ 35k
Correlate expression or polymorphisms with
phenotype