genentech final paper
Post on 15-Apr-2017
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Chemical control of recombination in drosophila for mapping neuronsPavel Morales, Sachin LethiUniversity of California, San Diego
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.
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.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.
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. Figure 2. Mapping of individual neurons using Hs-FLP method.
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 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 recombinationThe 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). Figure 3A. Flp-frt mechanism before recombination of sequence between frt sites.Figure 3B. Flp-frt mechanism after recombination and cleavage of sequence.
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 of individual cells can be mapped. Figure 4. DD fused with protein of interest.
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: -94C for 30 seconds, Annealing temperature of 45C for 30 seconds, Extension temperature of 68C 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.Figure 5. Agareose gel of colony pcr productFlp-DDplasmid1kbladder
UAS-flp-frt plasmid sequencingFlippase 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 plasmid. Figure 6. Plasmid sent back from sequencing
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 individual neurons will be monitored to see where their projection travels. Figure 7. Transgenic flies (orange eyes) vs. wildtype flies (white eyes).
Fly crossesTo 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 ResultsThrough 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