troubling news… …in genetics?
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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, - PowerPoint PPT PresentationTRANSCRIPT
Troubling News……in Genetics?
Jeff Young, BotanistBiology DepartmentWestern Washington [email protected]
Arabidopsis thalianaFunctional 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 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.
Reverse Genetics
FunctionSequence
DNA
Gene Disruption
Homologous Recombination
- or other -
Phenotype
GeneticsBiochemistryPhysiology
Phenotype Sequence FunctionClassical Genetics:
Homologous Recombination
• the replacement of a gene with an exogenous gene through equal crossing over,
Before
After
foreign DNA
Regions of Homology
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.
similarresults
The Question?
Can we identify a “genetic circuit” that affects behavior in mammals?
Trpc2 Knock Out
Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500
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
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 cellcontribution,
Chimeric founder - weaker ES cellContribution.
Chimeric mouse
Black/White Chimeric Example Cross and look for offspring with germ-line
transfection.
Trpc2 Knock Out Confirmation
SouthernWestern
Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500
in situ Hybridization
Science 22, February 2002Vol. 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?