1. Chemical control of recombination in drosophila for
mapping neurons
Pavel Morales, Sachin Lethi
University of California, San Diego
Abstract
Flp-frt recombination is an important tool for isolating and tracing neural
circuits. The projections of a cluster of neurons can be mapped by sparsely labeling
individual cells using flp-frt recombination. We propose to modify the flp-frt
recombination system to confer chemical control on flp-dependent recombination.
To do this, we plan to use the destabilizing domain (DD) technology. Linking DD
with any protein of interest destabilizes the fusion protein and marks it for
degradation. However, in the presence of a stabilizing ligand, trimethoprim, the
fusion protein is spared from degradation. We plan to fuse flippase to DD and thus
control its activity in a TMP dependent manner. This modification allows us to have
a greater temporal control of the recombination. Additionally, by varying TMP
dosage, we can control the sparseness of recombination in a cell population. We
plan to test this system using Drosophila melanogaster by making an flp-DD
transgenic fly and using it to map projections of individual neurons in the
Drosophila olfactory system.
Introduction/Background
The olfaction mechanism is something that has yet to be fully explained. The
brain creates a neural picture of what we experience from the external world and
then continues to control a behavioral response. A method to explaining this

2. mechanism is with the usage of the Drosphila, or fruit fly. In Drosphila, sensory hairs
are located in the third antennal segment and the maxillary palp, which recognize
odors. Projections from olfactory neurons that are located within the sensory hairs
are sent to glomeruli (cluster of neurons) in the antennal lobe of the brain. These
glomeruli are connected to higher olfactory centers by the projection neurons. It is
known that projection neurons express both distinctive and common receptor
genes, which allows for expression of a private and public specificity. In cases of
projection neurons that express the same receptor, only one or two glomeruli
within the antennal lobe are targets for the projection of these neurons. Hence, in
the antennal lobe odor receptor activity is shown as a topographic map. Receptors
elicit patterns of activity in the antennal lobe, which are communicated by higher
sensory centers to allow for the identification of olfactory information that is
needed for behavior responses. Thus, and understanding of the neural circuits that
translates odor recognition into specific behavioral responses is required.
Figure 1. A map
of all the neurons
and parts of the
brain involved in
the olfactory
system. Lateral
Horn mapped
GFP and
Antennal Lobe
mapped with
YFP.

3. Mapping out the neural circuits involved in olfaction. On a past research done
by Dr. Jing Wang and his collaborators, Spatial Representation of the Glomerular
Map in the Drosophila Protocerebrum, performed experiments that allowed the
projection of projection neurons that connect from glomeruli to higher olfactory
centers, mushroom bodies and protocerebrum, to be visualized. In his techniques,
Dr. Wang used the flippase (flp)-frt mechanism with heat shock to label individual
projection neurons using a CD8-GFP reporter.
However, this heat shock method raises potential problems for the fruit fly from the
high temperatures they have to endure during the experimentations. Drosophila
have a high olfactory sensitivity, which leads to behavior changes, they start to smell
differently. High temperatures also affect synaptic physiology where neural
transmitters start being released at different speeds, which affects the behavior of
the fly. My project investigates a different method of mapping neuron circuits
Figure 2.
Mapping of
individual
neurons using
Hs-FLP method.

4. without the usage of heat shock and avoiding the problems associated with it, by
instead using the flp-frt recombination mechanism with destabilizing domains.
Flp-frt recombination
The flp-frt recombination mechanism requires two specific sites, flippase
recognition target sites, which flippase binds to and recombines the sequence
between the sites in reverse orientation. Thus, cleaving the sequence between the
two sites. Controlling the orientation of frt sites allows us to completely remove the
sequence located between the two sites by making them have the same orientation.
This tool of removing the sequences between the sites is useful in that it can be used
as an identifier of flippase, in that a stop codon can lie between the frt sites (3A).
Flippase recombines the sequence between the frt sites then the stop codon gets
removed, transcription continues downstream of the second frt site (3B).
We design a construct that has GFP located past the frt sites that will help to
identify flippase activity. In order to map a projection of cluster of neurons,
individual cells need to be sparsely labeled, which is done by modifying the flp-frt
recombination system to confer chemical control on flp-dependent recombination
using destabilizing domains. Destabilizing domains (DDs) can be used to degrade
Figure 3A. Flp-frt
mechanism before
recombination of sequence
between frt sites.
Figure 3B. Flp-frt
mechanism after
recombination and cleavage
of sequence.

5. specific proteins that lack the stabilizing ligand. In the absence of the ligand DD are
degraded by the 26S proteasome, resulting in degradation of the protein of interest
that was fused alongside the DD. However, when a high-affinity ligand is added DD
stabilizes rapidly. When small amounts of ligand are present DD becomes
destabilized only in few individual cells, thus, GFP being expressed only in those few
individual cells. When GFP is expressed sparsely in a cluster of cells, the projection
of individual cells can be mapped.
Methods
Cloning plasmid construct
To design the desired UAS-flp-DD construct, two different plasmids, which
contain different components to the desired construct, were used. Plasmid 1
contained the Flippase gene. Plasmid 2 contained DD.
1. Forward and reverse primers will be designed that contain restriction
enzyme sites (used later for ligation) along with complementary sequence to
flp gene.
2. The optimal temperature for primers in PCR will be checked.
3. Followed by the amplification of flp gene using PCR.
Figure 4. DD fused
with protein of
interest.

6.  PCR Cycle: -94°C for 30 seconds, Annealing temperature of 45°C for 30
seconds, Extension temperature of 68°C for 1.5 minutes.
 PCR reagents (will be ran in agarose gel after completion to check for
correct amplification of flp gene): Standard Buffer, Forward and Reverse
primers, DNA, DnTPs, Polymerase, and Water.
4. DD vector cut with the same restriction enzyme that the primers contained
the restriction site to create sticky ends.
 Will be run in gel to verify if the vector was cut correctly with the
restriction enzymes.
 Measured concentration of both flp insert and DD vector for ligating
purposes.
5. Ligation will be setup for flp gene and DD plasmids.
6. Transformation- growing bacteria with UAS-flp-DD plasmid in liquid LB
media.
 E.coli will be used as competent cells to grow plasmid in.
 The E.coli will be let to grow in LB plates, which contain the ampicillin
antibody.
 Only bacteria that take in the plasmid that contains the ampicillin
resistance will grow.
7. Colony PCR
 To verify which of the colonies that grew in the LB plates took in the
correct plasmid.

7.  The sticky ends of DD vector can join together during ligation set up; it is
likely that bacteria take in this plasmid, rather than the one with the flp
insert.
 Check which of the colonies that grew contain the correct plasmid.
8. PCR product is run through an agarose gel, and then viewed under UV light to
spot out the bands of DNA on the gel.
 The bands containing flp plasmid are cut out of agarose gel to be purified.
UAS-flp-frt plasmid sequencing
Flippase gene was successfully fused to DD plasmid as shown by the gel
shown in figure 5. Figure 6 shows the plasmid that was sent back from sequencing;
flippase (green) next to DD (red). The plasmid also contains ampicillin resistance (in
yellow), origin of replication (grey), and the mini white gene (pink). Flies carrying
the mini-white gene show a different eye color other than white, that range from
pale yellow to red, depending on the positioning of the insert. This allows for
differentiation of which flies took in the UAS-flp-DD plasmid after fly injection of
Figure 5.
Agareose gel
of colony pcr
product
1kb
ladder
Flp-DD
plasmid

8. plasmid.
Transfecting Drosophila
Verification of the UAS-flp-DD construct will lead to sending it off for the
transfecting of the plasmid to Drosophila. Transfected flies will show orange eye
color as opposed to white-eye color flies that were unsuccessfully transfected with
the plasmid. Fruit flies with the UAS-flp-DD genome will be sent back. At this point
the fruit flies will be fed trimethoprim, the stabilizing ligand of DD. GFP tracking of
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
Figure 6.
Plasmid
sent back
from
sequencing

9. individual neurons will be monitored to see where their projection travels.
Fly crosses
To complete the transgenic fly needed for flp-frt recombination to DD, we
crossed UAS-flp-frt transgenic fly to two other transgenic flies. One of the other flies
contained the Gal4 line, which binds to the enhance UAS (Upstream Activation
Sequence) to activate gene transcription. The other fly contained the frt sites, stop
codon located between them, and GFP. The progeny of these three crosses gave us
the desired transgenic fly that allowed for experimentation.
Predicted Results
Through chemical control of flippase gene and GFP expression, mapping of
individual cell projections will be drawn. The labeling of neurons is directly
correlated to the amount of ligand present in the Drosophila’s system. Figure shows
the number of neurons that are green-fluorescently labeled when large amounts of
ligand are fed (top illustration), where all neurons have the green fluorescence.
Once the amount of ligand starts decreasing, so does the number of neurons
expressing green fluorescence. Until the amount of ligand decreases low enough for
Figure 7. Transgenic
flies (orange eyes)
vs. wildtype flies
(white eyes).

10. only one, or very few, neurons to have green fluorescence expression (bottom
illustration).
Through these results neurons are singled out from a group of neurons
whose projections to higher olfactory centers are uncertain.
Figure 8.

11. References
Ukrae, Cho. “Rapid and Tunable Control of Protein Stability in Caenorhabditis
elegans Using a Small Molecule.” PLOS ONE. August 2013. Volume 8. Issue 8.
Wong, Allan. “Spatial Representation of the Glomerular Map in the Drosophila
Protocerebrum.” Cell, Vol. 109, 229-241, April 19, 2002.