1. Neurobiology of Learning and Memory
Prof. Stephan Anagnostaras
Lecture 9: Molecular-Genetic Approaches to Learning & Memory

2. CamKIIa and CREB
are required for LTP

3. Synaptic Kinase activation Nuclear CREB activation
Mechanisms of Learning and Memory
NMDA
Synaptic Kinase activation D1
AMPA A Ca
Cyclase
cAMP
CaMKII
PDE
CREB
Nuclear CREB activation

4. 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

5. 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)

6. In vitro mutagenesis of a cloned gene
Gene knockout and
transgenic techniques
usually involve mutagenesis
of cloned genes prior to
transfer into the organism

7. Production of transgenic mice
Confusing example is a dominant negative transgene
(like Mayford & Kandel), not a knockout but a lot like a knockout

8. Creation of mice embryonic stem (ES) cells carrying a knockout mutation

9. Gene knockout in mice

12. •Disrupts autophosphorylation of Threonine at 286
Disruption of CamKIIa by T286A point mutation disrupts LTP and spatial memory
•Disrupts autophosphorylation of Threonine at 286
T=Threonine (ACT, ACC, ACA)
A=Alanine (GCT, GCC, GCA,GCG)
Unable to switch to
Ca-Calmodulin independent state
Giese et al., Science,1998

13. Cell-type-specific gene knockouts in mice: Cre-Lox technique Spatial and/or temporal control of deletion

14. Tsien et al., Cell, 1996: CA1 specific NMDAR1 knockout

15. CA1 DG
Tsien et al.,
Cell, 1996

16. McHugh et al., Cell, 1996:
Place cells in CA1 NMDAR KO
Abnormally large place fields
Low coherent firing

17. An Temporally controlled inducible CREB Repressor: The LBD Fusion Approach
Kida, Josselyn et al., 2002
LBD
CREBr
Heat Shock
Protein
Tamoxifen
CREBr
LBD
CREBr
LBD
Cytoplasm
Nucleus

18. 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)

19. Josselyn et al., 2002 - temporal control of the CREB repressor
Fear conditioning

20. Tetracycline-regulated expression of CamKIIa-Asp286 (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

21. Tet-Off System

22. 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

23. 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