1. Troubling News…
…in Genetics?

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

3. General Odor
Reception

4. Pheromone Reception
VNO surgical removal removes pheromone response.

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

6. 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.

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

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

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

10. 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

11. tail

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

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

14. Trpc2 Knock Out Science 22, February 2002
Vol no. 5559, pp

15. 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).

16. 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.

17. 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.

18. Vasectomizing

19. Successfully transformed ES cells are injected into blastocysts

20. 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.

21. Implanting blastocysts

22. Implanting Blastocysts (cont.)

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

25. Cross and look for offspring with germ-line transfection.
Chimeric mouse
Black/White Chimeric Example
Cross and look for offspring with germ-line transfection.

26. Trpc2 Knock Out Confirmation
Southern
Western
Science 22, February 2002
Vol no. 5559, pp

27. in situ Hybridization Science 22, February 2002
Vol no. 5559, pp

28. Heterozygous females are +/- like Mutants…

30. Female Intruder
Male Intruder
Heterozygous Female
Homozygous Female

31. Ultrasonic Sound

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