The overall goal of this procedure is to assess the effects of amyloid beta toxicity on neuronal function and survival. This is accomplished by first constructing transgenic C elegance worms expressing an amyloid beta peptide with a GFP reporter in a SE sensory neurons. Next, the behavioral consequence of expressing amyloid beta in a SE neurons is assessed by a chemotaxis assay.
The third step is to obtain primary cultures of a SE neurons and then treat cultured neurons with pharmacological agents such as Q-V-D-O-P-H to suppress the toxic effect of amyloid beta. Ultimately, results can show the effects of amyloid beta toxicity on a SE neurons function and survival through behavioral and pharmacological assays. This method can help answer key questions about neurodegeneration, such as elucidating specific cellular and molecular mechanisms.
Underlying neurotoxic effects of amyloid beta oligomers. Demonstrating the procedures will be Dr.Zing Dwan, a postdoc from my laboratory For the transformation, 2%agros injection pads are required. Place a drop of hot aros dissolved in water onto a glass cover slip, and quickly place a second cover slip on the drop and lightly tap it.
When making the injection mix, be sure to centri fut and transfer the top five microliters, free of debris and other contaminants to a fresh tube for injection load, injection mix and empty plasmid into an injection pipette by capillary action. Half a microliter is usually enough to inject 100 worms to widen the opening on the injection pipette rub its tip against the edge of a cover slip mounted on a glass slide in Halo carbon 700 oil. The quality of the opening is critical.
It is judged by the tip shape, and most importantly, by the flow rate, assessed by the flow and by the size of the bubbles forming at the pipette tip in response to injection pressure. Now place a drop of 700 hallow carbon oil on an injection pad and transfer several worms to the pad. Use a worm pick to push the worms down onto the pad until they adhere to the aros.
The worms should be oriented in rows with their ventral sides aligned. Avoid touching their heads. If after several attempts the worms fail to adhere to the pad, try a fresh pad or increase the agros thickness or concentration of the pad.
Now insert the pipette into the worm. First, move the stage to position the worm under the pipette second. Position the pipette next to the central core of the gonad.
Because its cytoplasm is shared by many germ cell nuclei, this improves the likelihood of the injected DNA being passed to the progeny. The pipette should lie almost parallel to the worm. Third, move the tip vertically towards the body until the skin is depressed.
Lastly, gently tap the manipulator to induce the tips insertion. Now inject the DNA solution. Apply pressure to the pipette until the gonad swells.
Then stop the flow and pull the worm off the pipette. Test the needles flow, and then inject the other gonads before moving on to the next worm. After the injections, add about 10 microliters of recovery buffer to the worms and wait for them to begin swimming.
Once swimming, add an equal volume of M nine buffer. When the worms resume swimming, continue adding M nine until the solution is mostly M nine. Now transfer each worm to a seated plate.
Collect GR worms in one milliliter of M nine buffer in a 50 milliliter falcon tube. Then using M nine buffer, wash the worms by repeated centrifugation and solution replacement until the M nine is clear after the wash. Next, add 10 volumes of basic hypochlorite solution to lice.
The pelleted worms incubate the worms at room temperature until about four fifths of them are broken, which typically takes 10 minutes. Monitor the reaction by taking aliquots to view with a microscope. Stop the reaction by adding an equal volume of sterile egg buffer.
Then collect the eggs and carcasses by centrifugation at 450 G for five minutes. Gently remove the supernatant. Add back an equal volume of egg buffer and centrifuge the tube again at 450 G for five minutes.
Repeat the procedure until the buffer is clear. Now incubate the eggs overnight in M nine buffer, and on the next day, seed them on standard NGM plates. Prepare several cubes of agar, roughly half a centimeter on a side.
Soak the agar chunks in a solution containing the desired attractant for two hours. Deposit an agar chunk in a 10 centimeter test plate in which the location of test spot and a control spot have been marked. Prepare five such plates per experiment.
Allow the plates to form a chemical gradient by leaving them overnight at 20 degrees Celsius the next day. Prior to the experiment, add 10 microliters of 20 millimolar sodium azide to each spot on the plates. This acts as an anesthetic.
Start the experiment by placing 20 age synchronized worms at the center of the plate, and then placing the plate in a 20 degree Celsius incubator for an hour. After an hour count the animals on the test over control spots and calculate the CI index. After lysing gr worms and cleaning the eggs and carcasses as described earlier, re suspend them in two milliliters of sterile egg buffer and add two milliliters of sterile 60%sucrose in egg buffer.
Then mix the tube by hand or by vortex until the eggs are completely resuspended. Centrifuge the resuspended eggs at 450 times G for 15 minutes. Then carefully collect the egg filled supernatant and discard the palate to remove the residual sucrose, resuspend the eggs in egg buffer and centrifuge them at 450 G for five minutes.
Gently collect and discard the supernatant and then repeat the cleaning process three times at the hood. Use sterile egg buffer containing one unit per milliliter of chitinase to resuspend the pelleted eggs. Allow the eggs to digest at room temperature for 30 minutes, and then start to monitor the reaction under an inverted cell culture microscope.
When 70 to 80%of the eggshells are digested, add five milliliters of complete L 15 culture media or CM 15. To isolate the cells, dissociate the cells so they do not form clumps by flowing them through a 27 gauge needle a few times. Now pass the solution through a syringe with a five micron filter.
Pellet the dissociated cell suspension by centrifugation at 450 G for 15 minutes. Then remove the supernatant and resuspend the pelleted cells in CM 15. To be sure the cells are dissociated, check the suspension under a microscope.
Finally, plate the dissociated cells on glass cover slips previously coated. With peanut lectin, the cells can be maintained at room temperature with normal air for more than two weeks. Using the transformation techniques described, a transgenic worm was constructed to express human a Beta 42 in the a SE neurons, neurons that detect water-soluble attractants such as biotin using the chemotaxis assay.
Seven day old transgenic worms were tested. Controls had no trouble finding biotin. By contrast, only a few worms expressing a beta 42 could find the attractant spot.
The chemotaxis index for biotin was 0.68 versus 0.12 for control and experiment worms respectively at three to four days. There was also a small but detectable drop in the index. A cultured A SER neuron at four days still expressing GFP after nine days of life.
However, GFP fluorescence disappeared and apoptosis was suspected to test this theory. Primary A SER neurons were prepared as described and supplemented with a broad spectrum caspase inhibitor. As expected, the treatment stopped the loss of fluorescence confirming that the expression of human A beta 42 triggers apoptosis in A SER neurons.
After watching this video, you should have a good understanding of how to construct the elegant transgenic worms, study them in behavioral essays, and how to obtain primary culture from Ambrose.
