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Troubling News…
…in Genetics?
Jeff Young, Botanist
Biology Department
Western Washington University
[email protected]
360-650-3638
Arabidopsis thaliana
Functional Genomics
Autoinhibitory H+-ATPase
Gene Family
AHAs
Pheromones
...Small volatile chemical
signals,
– function in communication
between animals,
– act much like hormones in
influencing physiology and
development.
General Odor
Reception
Pheromone Reception
VNO surgical removal
removes pheromone
response.
The Question(s)?
Can we genetically dissect behavioral
responses? Can we identify a “genetic
circuit” that affects behavior in mammals?
etc.?
Forward Genetics
Phenotype
map &
clone
Need mutant mice lacking
pheromone response(s).
Sequence
Function
Physiology, biochemistry,
more genetics, etc.
Complex biological phenomenon, such
as behavior, often lack clear, heritable
phenotypes.
Reverse Genetics
Sequence
Gene Disruption
Phenotype
DNA
Homologous
Recombination
- or other -
Genetics
Biochemistry
Physiology
Classical Genetics: Phenotype
Sequence
Function
Function
Homologous Recombination
• the replacement of a gene with an exogenous gene
through equal crossing over,
foreign DNA
Before
Regions of
Homology
After
The Question(s)?
Can we genetically dissect behavioral
responses? Yes
Can we genetically dissect behavioral
responses?
•
One region of mouse
chromosome 6 has a
cluster of 23 Vr1,
– 16 functional genes,
– 7 pseudogenes,
•
No other genes in the
region,
– removing this part of
the chromosome
should only affect V1r
associated biology.
V1r Gene Family Tree
tail
Behavior Analysis
What’s different about the KO mice?
Experiments derived from VNO surgery results.
similar
results
The Question?
Can we identify a “genetic circuit” that affects
behavior in mammals?
Trpc2 Knock Out
Science 22, February 2002
Vol. 295. no. 5559, pp. 1493 - 1500
Recombinant Mice
inserting the vector(s) via Homologous Recombination
• DNA is introduced into
embryonic stem (ES) cells via
electroporation,
– electrical shock makes
membrane leaky,
• ES cells that have undergone
homologous recombination are
identified by a selectable
marker(s),
•
and injected into a 4 day old
mouse embryo (blastocyst).
Electroporation
Embryonic Stem Cells
- harvested from the inner cell
mass of mouse blastocysts,
- grown in culture and retain
their full potential to produce all
the cells of the mature animal,
including its gametes.
Pseudopregnant Females
Vasectomized Males
• female mice can be tricked into thinking they are
pregnant,
– a mouse in estrus is mated with a vasectomized male
inducing pseudopregnancy,
• if eggs (transformed blastocysts) are implanted,
female will become truly pregnant and will give
birth to live transgenic offspring.
Vasectomizing
Successfully transformed ES
cells are injected into blastocysts
Implantation of Blastocysts
• The blastocysts are left to rest for a couple
of hours after cell implantation,
• Expanded blastocysts are transferred to the
uterine horn of a 2.5 dpc pseudopregnant
female,
• Viable pups are born.
Implanting blastocysts
Implanting Blastocysts (cont.)
Littermates
Black mouse no apparent ES cell
contribution,
Chimeric founder strong ES cell
contribution,
Chimeric founder weaker ES cell
Contribution.
Chimeric mouse
Black/White Chimeric Example
Cross and look for
offspring with germ-line
transfection.
Trpc2 Knock Out Confirmation
Southern
Western
Science 22, February 2002
Vol. 295. no. 5559, pp. 1493 - 1500
in situ Hybridization
Science 22, February 2002
Vol. 295. no. 5559, pp. 1493 - 1500
Heterozygous females are +/- like
Mutants…
Female
Intruder
Male Intruder
Heterozygous
Female
Homozygous
Female
Ultrasonic Sound
What do You Think?
Can we genetically dissect behavioral
responses?
Can we identify a “genetic circuit” that affects
behavior in mammals?
Questions?