Genentech Powerpoint

Chemical control of recombination in Drosophila for mapping neurons

Pavel MoralesGenentech

UC San Diego

Why is mapping neurons important?

• Helps understand the essential principles that control how neural circuits govern behaviors.

• Figuring out the anatomy of the brain on the cellular level.

Olfactory System• Helps explain the process neuron projections take when a particular

odor is smelt. A map of all the neurons and parts of the brain that are responsible for olfactory attraction and aversion is attained.

Sources: Spatial Representation of the Glomerular Map in the Drosophila Protocerebrum, 2002.

Antennal lobe

Lateral horn

Flp-frt recombination method• Flippase recognizes frt sites and “flips them” in

reverse orientation, thus cleaving sequence between the two frt sites.

Flippase

frt

frtstop GFP5’ 3’

frt GFP5’ 3’

Flp-frt recombination using heat shock

• Projections of single cells are mapped

Sources: Spatial Representation of the Glomerular Map in the Drosophila Protocerebrum, 2002.

Flp-frt recombination using heat shock

• Raises potential problems from the high temperatures that Drosphila have to endure during the experimentations.– Olfactory Sensitivity (behavior changes)– Synaptic physiology (neural transmitters start

being released at different speeds)

Destabilizing Domains (DDs)

• Method using DD requires for a chemical ligand to be present for a protein of interest to be expressed.

• In the absence of the ligand, DD becomes destabilized, resulting in degradation of the protein of interest that was fused alongside the DD.

Sources: Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule, 2013.

DDPOI DDPOI

ligand

degradation stable fusion

Destabilized GFP• Example of DD fused with GFP• On the right, shows neural mapping when ligand is present

(DD is stable), GFP is expressed.• Left, ligand absent = no GFP expression.

Promoter Gal4 UAS GFP DD

What is the aim of the project?

• Create UAS-flp-DD transgenic fly• Chemical control of flp-frt recombination

using destabilizing domains– Easier manipulation of experimental factors– No use of heat shock

Cloning

• 1) PCR – flp (add restriction sites)

• 2) Restriction digest (cuts flp out)

• 3) Ligation (flp into plasmid)

• 4) Transformation• 5) Sequencing

Sources: https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/cloning/

UAS-flp-DD plasmid

Flippase

DD

Sent for injection

• Takes 3-4 weeks

Red eyes indicate positive intake of plasmid

Fly Crosses

UAS flp DD UAS frt STOP frt GFPPromoter Gal4

x x

=UAS frt STOP frt GFP

UAS flp DD

Promoter Gal4

Predicted Results

Fed a small amount of ligand

Fed a large amount of ligand

Neurons

Neurons

Neurons

Acknowledgements

Wang Lab-Jing Wang (PI)-Sachin Sethi (Mentor)-Susy Kim (Mentor)

Thank you!

RE 2

RE 1

RE 1 RE 2

RE 1 RE 2

Flippase geneRE 1

siteRE 2 site

Vector contai-ning DD

Flippase gene

RE 1 site

RE 2 site