1. Chemical control of recombination
in Drosophila for mapping neurons
Pavel Morales
Genentech
UC San Diego

2. 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.

3. 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

4. Flp-frt recombination method
• Flippase recognizes frt sites and “flips them” in
reverse orientation, thus cleaving sequence between
the two frt sites.
Flippas
e
frt
frt
stop GFP
5’ 3’
frt
GFP
5’ 3’

5. Flp-frt recombination using heat shock
• Projections of single cells are mapped
Sources: Spatial Representation of the Glomerular Map in the Drosophila Protocerebrum, 2002.

6. 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)

7. 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

8. 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

9. 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

10. 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/

11. UAS-flp-DD plasmid
IVS 769..827
5X UAS 391..486
5X UAS 263..358
p10 2632..3308
AmpR 3727..4386
ColE1 origin 4484..5166
miniw+ 5522..9638
10xUAS-flp-dd
9638 bp
C-DD 2154..2630
Flippase 903..2125
Flippase 879..2147
Flippase 2126..2147
Flippase
DD

12. Sent for injection
• Takes 3-4 weeks
Red eyes indicate positive intake of plasmid

13. Fly Crosses
UAS flp DD UAS frt STOP frt GFPPromoter Gal4
x x
=
UAS frt STOP frt GFP
UAS flp DD
Promoter Gal4

14. Predicted Results
Fed a small amount of ligand
Fed a large amount of ligand
Neurons
Neurons
Neurons

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

16. Thank you!

17. RE 2
RE
1
RE
1
RE 2
RE
1 RE 2
Flippase
geneRE
1
site
RE
2
site
Vector
contai-
ning DD
Flippase gene
RE
1
site
RE
2
site