Transcript No Slide Title

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
map &
clone
Need mutant mice lacking
pheromone response(s).
Sequence
Function
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
• mice genes
( ~ 700),
(~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,
V1r Gene Family Tree
– mutations in one or even
many V1r genes may not
have readily observable
phenotypes.
Reverse Genetics
Sequence
Gene Disruption
Phenotype
DNA
Homologous
Recombination
- or other -
Genetics
Biochemistry
Physiology
Classical Genetics: Phenotype
Sequence
Function
Function
Homologous Recombination
• the replacement of a gene with an exogenous gene
through equal crossing over,
foreign DNA
Before
Regions of
Homology
After
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,
V1r Gene Family Tree
– removing this part of the
chromosome should only
affect V1r associated
biology.
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 neomycinr,
one with puromycinr
markers,
…double mutants
expressed both.
Hypoxantine-Aminopterin-Thymadine
Hprt: functional copy that
is present only after
Cre/lox recombination.
Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
Recombinant Mice
inserting 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
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 Example
Cross 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
Ga probe
No Morphological Defects
Figures 1e,f
Neurons, tissue, and organ development looks
normal in the KO mice.
tail
See Flash Animation
Behavior Analysis
What’s different about the KO mice?
Experiments derived from VNO surgery results.
Ligands/Electrophysiology
Figure 4
Serpentine receptors trigger
ion transport across the
plasma membrane,
...6-hydroxy-6methyl-3heptanone, 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.?
Why Cre, and what is
Hrpt and HAT?
What gets cut out and how do they know it?.
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.?”