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

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?

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.

Candidate Receptors G-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.

G-Protein Coupled Receptor

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

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

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

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

Chromosome Engineering Figure 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. Cre recombinase cuts out the ~600 kb Vr1a,b locus. Once the double mutant was made, a third transgene (Cre recombinase) was added to the cell via a plasmid.

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

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

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.

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

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

in situ Hybridization Figure 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 G  probe

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

tail

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

Ligands/Electrophysiology Figure 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.

What do You Think? Questions?

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

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

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

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.?”