Troubling News……in Genetics?

Jeff Young, BotanistBiology DepartmentWestern Washington Universityyoung@biol.wwu.edu360-650-3638

Arabidopsis thaliana

Functional Genomics

Autoinhibitory H+-ATPase Gene Family

AHAs

Pheromones

...Small volatile chemical signals,

– function in communication between animals,

– act much like hormones in influencing physiology and development.

General Odor Reception

Pheromone Reception

VNO surgical removal removes pheromone response.

The Question(s)?

Can we genetically dissect behavioral responses? Can we identify a “genetic

circuit” that affects behavior in mammals? etc.?

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.

Reverse Genetics

FunctionSequence

DNA

Gene Disruption

Homologous Recombination

- or other -

Phenotype

GeneticsBiochemistryPhysiology

Phenotype Sequence FunctionClassical Genetics:

Homologous Recombination

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

Before

After

foreign DNA

Regions of Homology

The Question(s)?

Can we genetically dissect behavioral responses? Yes

Can we genetically dissect behavioral responses?

• One region of mouse 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

tail

Behavior Analysis

What’s different about the KO mice?

Experiments derived from VNO surgery results.

similarresults

The Question?

Can we identify a “genetic circuit” that affects behavior in mammals?

Trpc2 Knock Out

Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500

Recombinant Miceinserting 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 cellcontribution,

Chimeric founder - weaker ES cellContribution.

Chimeric mouse

Black/White Chimeric Example Cross and look for offspring with germ-line

transfection.

Trpc2 Knock Out Confirmation

Southern

Western

Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500

in situ Hybridization

Science 22, February 2002Vol. 295. no. 5559, pp. 1493 - 1500

Heterozygous females are +/- like Mutants…

Female Intruder

Male Intruder

Heterozygous Female

Homozygous Female

Ultrasonic Sound

What do You Think?