Troubling News… …in Genetics?

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 Function map & clone Function Phenotype Sequence Physiology, biochemistry, more genetics, etc. Need mutant mice lacking pheromone response(s). Complex biological phenomenon, such as behavior, often lack clear, heritable phenotypes.

Reverse Genetics Function Function Genetics Biochemistry Physiology Sequence DNA Gene Disruption Homologous Recombination - or other - Phenotype Genetics Biochemistry Physiology Phenotype Sequence Function Classical Genetics:

Homologous Recombination the replacement of a gene with an exogenous gene through equal crossing over, foreign DNA Before Regions of Homology After

Can we genetically dissect behavioral responses? Yes 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? similar results What’s different about the KO mice? Experiments derived from VNO surgery results.

Can we identify a “genetic circuit” that affects behavior in mammals? 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, Electroporation ES cells that have undergone homologous recombination are identified by a selectable marker(s), and injected into a 4 day old mouse embryo (blastocyst).

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.

Cross and look for offspring with germ-line transfection. 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?