The overall goal of the following experiment is to refine GAL four expression through use of tissue specific lipase with GAL 80 converting tools. This is achieved by generating a collection of enhancer trap Philippe transgenic fly lines and determining the CNS expression pattern of lipase in each line. Using A GFP reporter IMMUNOCHEMISTRY and GFP reports can differentiate GL from neuronal expression to intersect GAL four expression in lines of interest ET flip lines across the flies containing a GAL 80 converting tool in the progeny.
Clonal analysis can be used to analyze the refinement of GAL four expression, such as in the eye. The main advantage of this technique over existing methods is that enhancer track play-based lines along with GAL 80 converting tools can be used with a fast collection of GAL four alliance already available in the fly community. Additionally, this concept can be expanded to other GALI four UAS emerging model organisms such as zebrafish.
This method can help answer key questions in mapping neural circuits underlying behavior. Although this protocol demonstrates the use of flip base in a subset of photoreceptor cells, the intersectional method can be applied to analysis of other tissues, including non neuronal tissues. As more enhancer trap lipase lines are characterized.
Gout four US is an important technique widely used by fly biologists. One caveat of most GAL four lines is that expressed too broadly to be used for brain behavior mapping using the finger methods. It's not possible to fine tune GAL four expression in T-shirts of interest Before using an ET flip transgene to partially restrict the expression of GAL four expression lines.
It's first helpful to know the expression pattern of ET flip x two transgene in late stage developing lava and in adults to visualize flip a's expression In CNS set up a cross between males with the ET t flip X two transgene of interest and virgin females harboring A GFP reporter for et flip expression from the progeny. Collect wandering third instar F1 larvae for dissection. Place them into a clean S guard dish and remove any debris with a paintbrush and rinse the dish once with one times PBS.
Now fill the dish with ice cold one times PBS to anesthetize the larvae to access the CNS of a lava hold its mouth hook with a pair of forceps. Grab the cuticle near the extending anterior spherical and peel it back. The CNS stays attached to the mouth hook along with some other structures.
Now with forceps, remove all those excess structures surrounding the CNS as you dissect the CNS. Transfer them individually into a three world Pyrex dish filled with PBS. Once all the CNS are dissected, fix them by fully submerging them in 200 microliters of 4%PFA on ice.
If a CNS floats, it has not been fully cleaned of all the surrounding tissue. Should this occur, use a pair of forceps to res submerge the CNS into the fixative after fixing for 15 minutes, wash the tissue with ice cold PB S3 times and then wash the tissue three more times in ice cold PBT. The tissue is now ready to be imaged or labeled by antibodies or stored in PBT at four degrees Celsius for later.
Staining from the same cross used to make larvae collect F1 adults prior to dissection. Submerged five to 10 flies and chilled 95%ethanol for 30 seconds to remove the wax on the surface of the cuticle. To remove the ethanol, wash the adults three times with ice cold one times PBS and after the third wash, transfer one of them to a 35 millimeter sil guard dish filled with ice cold one times PBS to prepare an adult for dissection.
Place it ventral side up and insert a minion pin midway through the abdomen. Begin dissecting by using forceps to remove the legs. Then remove the head cuticle by securing the head at the props and simultaneously peeling the cuticle from underneath the eye towards the top of the head.
Carefully using sharpened forceps, remove, track your tissues surrounding the brain because track your tissues have strong autofluorescence and it makes the brain float during fixing. The brain is only part of the adult CNS. The other part is the ventral nervous cord or VNC in the thorax and it must also be dissected.
First, remove the thoracic cuticle by securing the fly at the base of the hind leg and gently pulling off the thoracic cuticle up to the base of the neck. Second, to expose the VNC gently spread the two halves of the thoracic cuticle. Third, remove the abdomen from the thorax by securing at the base of the second leg and pulling away the abdomen from near the first abdominal stir night.
Fourth, clean the VNC by securing at the humeral area and gently tearing off the tissue surrounding the VNC. Tear apart the ring of connective tissue surrounding the cervical connective and continue removing excess tissue as you collect the brain. And VNC transfer them as pears into a nine world Pyrex dish filled with chilled one times PBS kept on ice.
To fix the adult tissues, submerge them in 4%PFA on ice for 15 minutes. Follow the bath with three ice cold PBS washes and three PBT washers. The tissue is now ready to be imaged or labeled by antibodies to analyze transgene expression with accuracy.
It is useful to label the CNS with neuronal or glial markers to provide a frame of reference begin by incubating the tissue at four degrees Celsius overnight with antibodies to either neurons or glia the next day. Wash the preparation five times with PBT and incubate with the fluorescent secondary antibody at room temperature for two hours. Wrap the plate and foil to prevent photobleaching of the antibody when the incubation is over.
Wash the preparation with PBS and whole mount the preparation to a slide. Use an OSHA plastic ring to prevent the tissue from being crushed by the cover slip. The nervous system is now ready to be examined for GFP and LL expression patterns under a fluorescent microscope.
Typically three to five replicates are examined to determine the reproducibility of the flip expression pattern. The expression pattern of flip in the flip, a line can be used along with other transgenes to change how the GAL four repressor GAL 80 is expressed. Changing the GAL 80 repressor expression is an effective method for making changes.
In GAL four expression, one method is to flip out GAL 80 expression with the transgene GAL 80 expression inactivates GAL four, which is otherwise driving eye degeneration throughout the eye. The ET flip line hash 6 88 A is used here to evaluate the effectiveness of the GAL 80 converting tool in a subset of photoreceptors in the F1 progeny, compare the eye phenotype to the eye phenotype of the parental strains. Any appearance of eye deformity or depigmentation confirms that the flip expression pattern is present in the photoreceptors and GAL 80 flipout was successful.
A complimentary non-redundant strategy is to flip in GAL 80 expression using the same flip line of interest. This strategy is very similar to the other, but now ID pigmentation and degeneration in the F1 progeny is suppressed. In flip X two.
Expressing photoreceptors Once mastered dissection of the CNS can be done in approximately five minutes for adults and one minute for larvae if it's performed properly. While attempting this procedure, it is important to use sharp forceps to remove the trachea and tissue surrounding both adult and Libre C and Ss.It is important to use a fixative free dish for dissection. Heat shock flip is currently used to generate random clones.
In contrast, enhancer trap flip is allows one to generate reproducible clones, making morphological and behavioral analysis easier. Another strength of the finger method is that one could further restrict GAL four expression patterns by using additional anan trap flip based lines and other gal lines. A weakness of the tubulin stop GAL 80 configuration is that certain combinations of GA four and UAS effectors such as LAG four and U-A-S-K-I-R are not compatible because they will kill the flies.
This problem can be overcome easily by using a conditional UAS effector, such as UAS Shiri Ts, which allows a behavior to be examined at restrictive temperatures As expected. Not all enhancer trap lipase lines will express high enough levels of lipase to be useful. Nonetheless, the finger technique described here provides a new approach for drosophila researchers to refine mosaic analysis either for development circuits or behavior.
