1. Troubling News… …in Genetics?

2. Jeff Young, Botanist Biology Department Western Washington University young@biol.wwu.edu 360-650-3638 Arabidopsis thaliana Functional Genomics Autoinhibitory H + -ATPase Gene Family AHAs

3. Pheromones...Small volatile chemical signals, –function in communication between animals, –act much like hormones in influencing physiology and development.

4. General Odor Reception

5. Pheromone Reception VNO surgical removal removes pheromone response.

6. The Question(s)? Can we genetically dissect behavioral responses? Can we identify a “genetic circuit” that affects behavior in mammals? etc.?

7. 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.

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

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

10. The Question(s)? Can we genetically dissect behavioral responses? Yes

11. 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

12. tail

13. Behavior Analysis What’s different about the KO mice? Experiments derived from VNO surgery results. similar results

14. The Question? Can we identify a “genetic circuit” that affects behavior in mammals?

15. Trpc2 Knock Out Science 22, February 2002 Vol. 295. no. 5559, pp. 1493 - 1500

16. Recombinant Mice inserting 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

17. 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.

18. 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.

19. Vasectomizing

20. Successfully transformed ES cells are injected into blastocysts

21. 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.

22. Implanting blastocysts

23. Implanting Blastocysts (cont.)

25. Littermates Black mouse - no apparent ES cell contribution, Chimeric founder - strong ES cell contribution, Chimeric founder - weaker ES cell Contribution.

26. Chimeric mouse Black/White Chimeric ExampleCross and look for offspring with germ-line transfection.

27. Trpc2 Knock Out Confirmation Southern Western Science 22, February 2002 Vol. 295. no. 5559, pp. 1493 - 1500

28. in situ Hybridization Science 22, February 2002 Vol. 295. no. 5559, pp. 1493 - 1500

29. Heterozygous females are +/- like Mutants…

31. Female Intruder Male Intruder Heterozygous Female Homozygous Female

32. Ultrasonic Sound

33. What do You Think? Can we genetically dissect behavioral responses? Can we identify a “genetic circuit” that affects behavior in mammals? Questions?