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Troubling News… …in Genetics?

Troubling News… …in Genetics?. Genetics and Behavior Reverse Genetic Analysis. Pheromones. ...Small volatile chemical signals, function in communication between animals, act much like hormones in influencing physiology and development. . General Odor Reception. Pheromone Reception.

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Troubling News… …in Genetics?

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  1. Troubling News… …in Genetics?

  2. Genetics and Behavior Reverse Genetic Analysis

  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

  6. The Question(s)? • What genes (especially receptors) are involved in pheromone responses in mice? • How do those genes affect behavior? • What compounds activate the protein products of those genes?

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

  8. Candidate ReceptorsG-Protein Coupled • Seven-transmembrane (serpentine) receptors, • olfactory receptors, • humans genes ( ~ 700), • mice genes (~1,400), • Co-expression of many different receptor genes allows the organism to sense complex mixtures of stimuli.

  9. G-Protein Coupled Receptor

  10. Why no Forward Genetic Phenotypes?Figure 1a • DNA array data (and other data) indicate that the V1r and V2r family of genes are involved in pheromone responses, • ~137 genes are in the V1r gene family, • mutations in one or even many V1r genes may not have readily observable phenotypes. V1r Gene Family Tree

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

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

  13. Why V1rb and V1ra Genes?Figure 1a • One region of 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

  14. Once the double mutant was made, a third transgene (Cre recombinase) was added to the cell via a plasmid. Cre recombinase cuts out the ~600 kb Vr1a,b locus. Chromosome EngineeringFigure 1b Two transgenes are inserted into the mouse genome, …one at each end of the V1ra,b multi-gene locus, ...each with a loxp sequence.

  15. Hprt: functional copy that is present only after Cre/lox recombination. Hypoxantine-Aminopterin-Thymadine Selectable MarkersFigure 1b Two transgenes are inserted into an hprt deficient mouse genome, …one with neomycinr, one with puromycinr markers, …double mutants expressed both. Hypoxanthine-guanine phosphoribosyltransferase (HPRT)

  16. Recombinant Miceinserting the vector(s) via Homologous Recombination • DNA is introduced into embryonic stem (ES) cells via electroporation, • electrical shock makes membrane leaky, • ES cells that have undergone homologous recombination are identified by a selectable marker(s), • and injected into a 4 day old mouse embryo (blastocyst). 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.)

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

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

  26. Knockout ConfirmationFigure 1c • Genomic DNA from the mice was blotted onto membrane (target), • Probed with VR1a,b genes (probes).

  27. in situ HybridizationFigure 1d Vomeronasal epithelium tissue, fixed, and then hybridized with V1rb1 probe. Vomeronasal epithelium tissue, fixed, and then hybridized with V1r gene probe (outside of the deletion). Vomeronasal epithelium tissue, fixed, and then hybridized with Ga probe

  28. No Morphological DefectsFigures 1e,f Neurons, tissue, and organ development looks normal in the KO mice.

  29. tail

  30. See Flash Animation Behavior Analysis What’s different about the KO mice? Experiments derived from VNO surgery results.

  31. Ligands/ElectrophysiologyFigure 4 Serpentine receptors trigger ion transport across the plasma membrane, ...6-hydroxy-6methyl-3-heptanone, n-pental acetate and isobutylamine appear to have lost their efficacy in the KO mice.

  32. What do You Think? Questions?

  33. Possible Exam Figure 1a • What are V1r genes • Why V1r genes? • Why V1ra,b family members? V1r Gene Family Tree

  34. Why Cre, and what is Hrpt and HAT? What gets cut out and how do they know it?. Chromosome EngineeringFigure 1b Why two Homologous Recombination constructs? where?, includes what DNA/Genes, etc.?

  35. Why is Table 1 crucial to the paper? Compare these results with those reported in Figure 2?

  36. Friday • Review, • Please do not ask me to simply provide the answers, • As in, “What do you want for Figure?” • Ask good questions, and I will readily provide you answers, • As in, “In Figure ?, why, how, what, when, etc.?”

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