Download Molecular Genetics - Temple University

Molecular Biological and Genetic
Techniques for Studying Learning
and Memory
Thomas Gould, Ph.D.
Department of Psychology
Temple University
Transgenic Techniques
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Inserts a novel gene into genome
Developed in the early 1980s by John Gordon and by Ralph
Brinster and Richard Palmiter and their co-workers.
Although all of the cells in the body contain an identical set of
genes, some genes are active in only one or a few tissues.
The two main parts of a gene are the regulatory region and the
protein-coding region.
When the right combination of proteins binds to specific sites
along the DNA in the regulatory region, the gene is switched on,
and the protein-coding region becomes active.
Transgenic Techniques
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Make your DNA
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Using recombinant DNA methods, build molecules of DNA
containing the promoter and structural gene you desire
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Insert into plasmid DNA to copy
 Cloning
Transform ES cells in culture
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Expose the cultured cells to the DNA to allow incorporation
Select for successfully transformed cells
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neor (a gene that encodes an enzyme that inactivates the antibiotic
neomycin and its relatives, like the drug G418, which is lethal to
mammalian cells) is part of the vector
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Expose embyronic stem cells to G418
Inject surviving cells into the inner cell mass (ICM) of mouse
blastocysts.
Transgenic Techniques
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Embryo transfer
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Transfer the embryos into her uterus.
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No more than one-third will implant successfully
Test offspring
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Prepare a pseudopregnant mouse (by mating a female mouse with a
vasectomized male - stimulus of mating elicits the hormonal changes needed to
make her uterus receptive)
Remove a small piece of tissue from the tail and examine its DNA for the
desired gene
No more than 10-20% will have it, and they will be heterozygous for the gene
Establish a transgenic strain
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Mate two heterozygous mice and screen their offspring for the 1:4 that will be
homozygous for the transgene
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Or create dominant transgene
Mating these will found the transgenic strain.
Knockout Mice
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DNA that has been mutated is injected into
embryonic stem cells in cell culture
Stem cells are injected into blastocysts that will
incorporate the cells
Cells need to be incorporated into the gametes to
be useful (low probability)
Mice born with this mutation are called chimeras
and have one copy of the mutated DNA
Chimeras are crossbred producing ¼ offspring
with two copies of mutated gene
Cerebellar Circuit
Eye-Blink Circuit
Parallel Fibers
Climbing Fibers
Cerebellar Cortex
Granule
Cells
Cerebellar Interpositus
Nucleus
Inferior
Olive
Mossy Fibers
Red Nucleus
Lateral
Pontine
Nucleus
(CS)
Trigeminal
Nucleus (US)
Abducens Nucleus
Reticular
Formation
Genetic Analysis of Cerebellar
Plasticity
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Current goals
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Use genetic manipulation to examine effects of pre- and
postsynaptic up and down regulation of PKA and
CREB at the granule cell Purkinje cell synapse on
classical conditioning of the eye-blink reflex and on
cerebellar LTP and LTD
Use the tetracycline system to temporally control gene
expression
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Examine Developmental issues
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Study acquisition vs extinction
Use Genetic Manipulation to examine age-related
changes in learning and memory
Temporal Control of Transgene
Expression
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Tetracycline Responsive Transciptional Activator
(tTA) is used to temporally control transgene
expression
tTA stimulates gene expression from its cognate
promoter
Doxycycline inhibits promoter activity
Spatial and Temporal Control of
Transgene Expression
Double Transgenic Mice
Region Specific Promoter
+
tTA responsive Promoter
tTA-Gene
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Effector Gene
Effector Genes
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R(AB) transgene is the dominant negative form of
the regulatory subunit of PKA
C(QR) transgene has a mutation in the catalytic
subunit which up regulates PKA
ICER (inducible cAMP early repressor) transgene
down regulates CREB via transcription factor
repression
CREBY/F transgene promotes CREB gene
expression via constitutive phosphorylation of a
mutant polypeptide at Ser 133.
Region Specificity
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6 promoter from the type A gammaaminobutyric acid receptor alpha6-subunit gene is
only expressed in cerebellar granule cells
L7 promoter is from a Purkinje cell-specific gene.
Spatial and Temporal Control of PKA
Expression in Cerebellar Granule Cells
Double Transgenic Mice
6 Promoter
+
tTA responsive Promoter
tTA-Gene
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R(AB) Transgene
Spatial and Temporal Control of PKA
Expression in Cerebellar Purkinje Cells
Double Transgenic Mice
L7 Promoter
+
tTA responsive Promoter
tTA-Gene
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R(AB) Transgene
Microarrays
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Method of examining changes in gene
expression associated with event, drug, or
disease
Probing Microarrays
Delay Contextual Fear Conditioning
Training Day
Testing Day
Context - Shock US
Association
Hippocampal
Dependent
Test Freezing to
Context
Clicker CS - Shock
US Association
Hippocampal
Independent
Test Freezing to
CS in Altered
Context
CS = 30 sec white noise, US = 0.5 mA 2 sec shock, ITI 2 minutes, 2 trials
Duration of Enhancement
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Nicotine must be administered on training
and testing days for enhancement. Will
enhancement be seen in the absence of
nicotine at a second test?
Groups
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Nicotine pre-training and prior to testing at 24
hours; retest one week later with no nicotine
Saline pre-training and prior to testing at 24
hours; retest one week later with no nicotine
% Freezing
100
Nicotine Enhances Contextual Fear 1 Week
Post Injection
80
60
40
20
0
24 hr
1 week
0
0.05
0.125
0.25
Nicotine (mg/kg)
0.375
Nicotine Alters Gene Expression
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Long-term memory for contextual fear conditioning remained
enhanced at later retests in the absence of nicotine (Gould and
Higgins, 2003)
Long-term memory is thought to be stored in neurons as a result
of changes in gene expression induced by the activation of
intracellular signaling pathways (reviewed in Abel and Lattal,
2001)
Nicotine can activate cellular and molecular processes involved
in the chain of events linking synaptic activity to gene
expression (Berg and Conroy, 2002; Dajas-Bailador et al., 2002)
Use microarray analysis to determine if hippocampus-dependent
learning in the presence of nicotine results in a different pattern
of gene expression than learning in the absence of nicotine
Affymetrix Microarrays
6000 mouse genes per chip
Arrayed as oligonucleotides; 20 per
gene
Mismatch oligonucleotides used as
controls
Does nicotine alter gene expression
in the hippocampus during fear
conditioning?
Microarray Experimental Design
Train and test mice: nic/nic; sal/sal
Prepare mRNA from hippocampus, amygdala, prefrontal;
label cRNA prepared from mRNA; hybridize to mouse
Affymetrix microarray MGU74Av2
Compare gene expression levels; compare to “normal”
variance in these brain regions; compare to mice treated
with nicotine or saline and not conditioned
Confirm using real-time PCR
Nicotine and Saline Mean Hippocampi Log
Base 2 Expression Values
Each dot represents the
expression level of a single
probe set (Gene) on both
chips.
Dots outside the line of
(y=x) are outliers potentially
represent genes that affected
by the nicotine manipulation.
Manipulation does not affect
most of the genome. The are
about 20 genes that appear
affected between the
expression levels of 5-11-representing about 1.5 fold
changes.
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Changes In Gene Expression
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Differences between nicotine and saline arrays were
specific because there were no global changes in
average expression between the groups
20 Genes with Significantly Higher Expression in
Nicotine Group
3 Genes with Significantly Higher Expression in Saline
Group
Potential Genes of Interest
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Potassium voltage-gated channel, shaker-related subfamily,
beta member 1
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Centrin 2
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May be involved in modulating chromatin formation and
contribute to regulation of cell proliferation
Mitogen activated protein kinase 8
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Inhibitor of phospholipase a2
Nucleosome assembly protein 1-like 1
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Coding for calmodulin
Annexin A3
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Accessory potassium channel protein which modulates the activity
of the pore-forming alpha subunit & alters the functional properties
of kv1.1 and kv1.4
phosphorylating a number of transcription factors
Mitogen activated protein kinase 10
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phosphorylating a number of transcription factors
Conclusions
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Nicotine enhances hippocampus-dependent
versions of fear conditioning
Nicotine enhancement of fear conditioning
is long-lasting and this long-lasting memory
is expressed in the absence of nicotine
Nicotine administration during training and
initial testing is associated with an upregulation of genes that may have a role in
synaptic plasticity
Acknowledgements
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Transdisciplinary Tobacco Use Research
Center UPENN
American Federation of Aging Research
The PA Department of Health (TG)
Thanks
Gould Lab
Jennifer Davis
Mike Lewis
Olivia Rossebo
Dan Moore
Steve Higgins
Joel Lommock
Alla Kryss
Collaborators and Colleagues
Ted Abel, Ph.D. UPENN
Sheree Logue, Ph.D. Aventis Pharmaceutical
Jeanne Wehner, Ph.D. Univ Colorado
Diana Woodruff-Pak, Ph.D. Temple Univ