Chemical control of recombination in drosophila for mapping neurons

Pavel Morales, Sachin LethiUniversity 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

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.

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

Figure 2. Mapping of individual neurons using Hs-FLP method.

transmitters start being released at different speeds, which affects the behavior of

the fly. My project investigates a different method of mapping neuron circuits

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

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

Figure 3A. Flp-frt mechanism before recombination of sequence between frt sites.

Figure 3B. Flp-frt mechanism after recombination and cleavage of sequence.

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.

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.

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

1kbladder

Flp-DDplasmid

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

Figure 6. Plasmid sent back from sequencing

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

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.

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.