Download - Troubling News… …in Genetics?
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Troubling News……in Genetics?
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Pheromones
...Small volatile chemical signals,
– function in communication between animals,
– act much like hormones in influencing physiology and development.
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General Odor Reception
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Pheromone Reception
VNO surgical removal removes pheromone response.
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The Question(s)?
Can we genetically dissect behavioral responses? Can we identify a “genetic
circuit” that affects behavior in mammals? etc.?
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Forward Genetics
Phenotype Sequence Function
Need mutant mice lacking pheromone response(s).
map &clone
Physiology, biochemistry, more genetics, etc.
Complex biological phenomenon, such as behavior, often lack clear,
heritable phenotypes.
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Reverse Genetics
FunctionSequence
DNA
Gene Disruption
Homologous Recombination
- or other -
Phenotype
GeneticsBiochemistryPhysiology
Phenotype Sequence FunctionClassical Genetics:
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Homologous Recombination
• the replacement of a gene with an exogenous gene through equal crossing over,
Before
After
foreign DNA
Regions of Homology
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The Question(s)?
Can we genetically dissect behavioral responses? Yes
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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
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tail
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Behavior Analysis
What’s different about the KO mice?
Experiments derived from VNO surgery results.
similarresults
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The Question?
Can we identify a “genetic circuit” that affects behavior in mammals?
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Trpc2 Knock Out
Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500
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Recombinant Miceinserting the vector(s) via Homologous Recombination
• ES cells that have undergone homologous recombination are identified by a selectable marker(s),
• and injected into a 4 day old mouse embryo (blastocyst).
• DNA is introduced into embryonic stem (ES) cells via electroporation,
– electrical shock makes membrane leaky,
Electroporation
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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.
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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.
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Vasectomizing
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Successfully transformed ES cells are injected into blastocysts
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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.
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Implanting blastocysts
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Implanting Blastocysts (cont.)
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Littermates
Black mouse - no apparent ES cell contribution,
Chimeric founder - strong ES cellcontribution,
Chimeric founder - weaker ES cellContribution.
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Chimeric mouse
Black/White Chimeric Example Cross and look for offspring with germ-line
transfection.
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Trpc2 Knock Out Confirmation
Southern
Western
Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500
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in situ Hybridization
Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500
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Heterozygous females are +/- like Mutants…
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Female Intruder
Male Intruder
Heterozygous Female
Homozygous Female
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Ultrasonic Sound
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What do You Think?