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Vertebrate Genetics
Nirao Shah
Haploid facts of life
Human Life Cycle
Drive at 16 yrs
Vote, marry, shoot at 18 yrs
C2H5OH at 21 yrs
~5 days
~12 yrs
~7 days
~6 mths
~8 weeks
~39 weeks
Sex determination in the mouse
XX = female
XY = male
XO = female
XXY = male
What chromosome harbors the sex-determining locus?
Y chromosome
Single gene on Y, Sry, is the sex determining locus
Mid-gestation embryo, E9.5
Bi-potential gonad
Sry
Testes
Testosterone
Bipotential body & brain
Male phenotype
Default
Ovaries
Estrogen & Progesterone
Non-autonomous
Female phenotype
How know Sry is sex determining locus?
Is it sufficient?
Is it necessary?
Forward genetics: Phenotype to genotype
Reverse genetics: Genotype to phenotype
Why do forward genetics?
Why not forward genetics in vertebrates?
The mouse:
• is diploid
• is not transparent
• does not self-fertilize
Screen for the phenotype you want,
Unbiased, Saturation, Allelic series,
Time ($), Space ($), Unlikely to saturate,
Some problems not accessible (incl in
flies and worms)
How know Sry is sex determining locus?
Is it sufficient?
Is it necessary?
Sufficiency - drive transgene in female and masculinize
- rescue Sry null with transgene
Necessity
- knockout Sry (doesn’t work bc cannot recombine on Y)
- deletions in the population
Reverse genetics in the mouse
Transgene – Random insertion of DNA construct in the genome
reporters, toxins, RNAi, rescue, sufficiency, enhancer bash
Targeted manipulation –
homologous recombination mediated
aka gene targeting, knock-in, knock-out
Basic ingredients for a transgenic construct
Generating a mouse bearing the desired transgene
Fertilized eggs from donor WT females
Inject your DNA
Transfer injected eggs into WT recipient females
Genotype progeny (why?, how?)
Breed founder progeny harboring the transgene
Check progeny of founders for expression of your transgene
Why?
How?
Male pronuclear injection of transgene
Typically integrates into genome as a multi-copy insertion
No expression or mis-expression of your transgene:
• Inadequate promoter, lacking regulatory elements
• Position effect: local genomic context precludes correct expression
Solutions:
• Screen more progeny
• BAC transgenic
• Lentivirus transgene
• Gene targeting
BAC transgene
•
Bacterial artificial chromosome
•
~150 kb genomic insert (publicly available library spanning the genome)
•
BAC libraries fully annotated, order by phone
•
Random integration
•
Lower freq of position effect
•
Gene regulatory information usually within the BAC you ordered
•
Usually single copy insertion
What’s the construct?
GENSAT collection of BAC transgenics – BAC mouse by phone
(http://www.gensat.org/index.html)
BAC transgene
Chunks can break off during injection
Incomplete regulatory information – misexpression
(or more rarely, position effect)
Lentiviral transgene
• HIV backbone stripped of all viral genes (why?)
• Recombinant and replication incompetent (why?)
• Produced in tissue culture
• Just soak fertilized eggs in virus and transfer to female
• Position effect rare (Integration usually close/within active locus)
• Pan-tropic (rats, voles.....)
• Max insert size is 10 kb (why is this bad?)
• Integration usually close/within active locus
A genetic approach to understand how we recognize odors
We recognize ~ 103 odors
How does the nose detect so many odors?
How does the brain know what the nose detects?
The olfactory pathway into the brain
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Olfactory
nerves
Axel 1995
Forward genetic screens in flies/worms failed to identify odorant receptors.
Why?
Redundancy in receptors
Difficult behavioral screens
Cloning olfactory receptors
•
Membrane preps of nose show
•
This requires GTP
cAMP in presence of odorants
In the late 1980s, Linda Buck & Richard Axel reasoned that,
1. odorant receptors must be GPCRs
2. these GPCRs must be encoded by a gene family
3. these GPCRs must be restricted to the nose
Cloning olfactory receptors
Buck & Axel designed degenerate PCR primers based on known GPCRs
(PCR had just been invented!)
Performed RT-PCR on mRNA from nose
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Cloning olfactory receptors
*
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GPCRs encoded by a gene family in the nose
Cloning olfactory receptors
GPCRs encoded by a gene family in the nose
are not expressed elsewhere
Cloning olfactory receptors
~1300 odorant GPCRs encoded in the genome
~5% of coding genes in genome
Highly conserved across vertebrates
Flies and worms have unrelated GPCR families of odorant receptors.
Identified later from mining the sequenced genomes in mid-90s.
Worm GPCRs identified by Emily Troemel in Bargmann lab at UCSF.
How does the brain know which OR has been bound by an odorant?
~ 107 olfactory sensory neurons in mouse nose
~ 1300 ORs
Could have combinatorial expression, several defined ORs per neuron
Alternately, single OR gene per neuron.
• In situ hybridization (ISH) reveals sparse expression of individual ORs (0.1 - 0.5% neurons)
• ISH pattern additive (what does this mean?)
• Although seemingly stochastic, symmetric expression across midline
• Reproducible between individuals
Ressler; Vassar 1993
How does the brain know which OR has been bound by an odorant?
ORs: I7, F3, J7
Olfactory marker protein (OMP)
1 mm
Vassar 1993
How does the brain know which OR has been bound by an odorant?
OR: I7
OR: J7
OMP
OR: I7
Vassar 1993
How does the brain know which OR has been bound by an odorant?
Taken together, highly probable that each olf. sensory neuron expresses only one OR.
Eventually confirmed by single cell RT-PCR.
Serial dilution of dissociated MOE population reveals something astonishing:
An olf. sensory neuron expresses only one allele of the OR.
In other words, ORs are expressed mono-allelically.
The problem of determining which OR has been bound by odorant is
reduced to distinguishing which olf. sensory neuron has been activated.
Ressler; Vassar 1993
Chess 1994