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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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Presentation Transcript
slide1

Troubling News…

…in Genetics?

slide2

Genetics and Behavior

Reverse Genetic Analysis

pheromones
Pheromones

...Small volatile chemical signals,

  • function in communication between animals,
  • act much like hormones in influencing physiology and development.
slide4

General Odor

Reception

the question s
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

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.

candidate receptors g protein coupled
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.
why no forward genetic phenotypes figure 1a
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

Sequence

Gene Disruption

Phenotype

Homologous

Recombination

- or other -

DNA

Genetics

Biochemistry

Physiology

Function

Classical Genetics:

Phenotype

Sequence

Reverse Genetics

Function

homologous recombination

foreign DNA

Before

Regions of Homology

Homologous Recombination
  • the replacement of a gene with an exogenous gene through equal crossing over,

After

why v1rb and v1ra genes figure 1a
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

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.

selectable markers figure 1b

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)

recombinant mice inserting the vector s via homologous recombination
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

slide17

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
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.
implantation of 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.
littermates
Littermates

Black mouse -

no apparent ES cell

contribution,

Chimeric founder -

strong ES cell

contribution,

Chimeric founder -

weaker ES cell

Contribution.

chimeric mouse
Chimeric mouse

Black/White Chimeric Example

Cross and look for offspring with germ-line transfection.

knockout confirmation figure 1c
Knockout ConfirmationFigure 1c
  • Genomic DNA from the mice was blotted onto membrane (target),
  • Probed with VR1a,b genes (probes).
in situ hybridization figure 1d
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

no morphological defects figures 1e f
No Morphological DefectsFigures 1e,f

Neurons, tissue, and organ development looks normal in the KO mice.

behavior analysis

See Flash Animation

Behavior Analysis

What’s different about the KO mice?

Experiments derived from VNO surgery results.

ligands electrophysiology figure 4
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.

possible exam figure 1a
Possible Exam Figure 1a
  • What are V1r genes
  • Why V1r genes?
  • Why V1ra,b family members?

V1r Gene Family Tree

chromosome engineering figure 1b1

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.?

slide37

Why is Table 1 crucial to the paper?

Compare these results with those reported in Figure 2?

friday
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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