genentech powerpoint
TRANSCRIPT
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