Encyclopedia of Experiments: Biology
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Transcript
- To prepare for the injection, use a worm pick to lay a track of halocarbon oil on an agarose pad. Place worms in the oil and immobilize them by gently pushing them onto the agar pad. Ensure the oil fully covers the animals. It allows oxygen to get to the worms while preventing desiccation. Next, place the agarose pad on an injection microscope and position the needle perpendicular to the worm, targeting the distal end of a gonad arm.
Gently push the needle through the cuticle and inject the gonad arm with the nucleic acid or compound of interest. Each gonad arm contains a germline syncytium that will develop into individual oocytes. Material injected into the distal gonad will be incorporated into those oocytes and affect the next generation.
After injecting worms, replace the oil with M9 buffer. Once the worms start moving, transfer them to an NGM agar plate with bacterial food. In the following protocol, the gonad microinjection method is used to deliver a Cas9 complex to target and edit the dpy-10 gene.
- Begin with preparation of C. elegans and agarose pads as described in the text protocol. Place an agarose injection pad and cover slip onto a dissection scope. Use a worm pick to lay a small track of halocarbon oil along one edge of the pad. Then, use the worm pick coated in oil to lift several worms off the NG agar plate and into the track of oil. With a fine hair attached to a pipette, such as an eyelash or cat whisker, position the worms in parallel gently pushing the worms into the agarose pad. Until comfortable with the microinjection procedure, only mount and inject one worm at a time.
Once in position and attached to the pad, overlay the worms with another few drops of halocarbon oil from the tip of the worm pick. Place the cover slip with the mounted worms onto the injection microscope. Under a low magnification, position the worms perpendicular to the injection needle, which has been filled with the RNP solution, as described in the text protocol.
Switch to a high magnification and reposition the needle adjacent to the gonad arm, corresponding to the region near the nuclei in mid- to late- pachytene. Using the micromanipulator, move the needle against the worm, depressing the cuticle slightly. Then, with one hand, tap the side of the microscope stage to jolt the needle through the cuticle. Depress the injection pedal or button, slowly fill to gonad arm with the injection mix, and remove the needle. Repeat the step with the other gonad arm.
Once the worms are injected, remove the coverslip in the agarose pad and place it under a dissecting microscope. Using a pulled capillary pipette, displace the oil from the worms by pipetting an M9 buffer over them. Perform this treatment to release the worms from the agar. After 10 minutes, when the worms are thrashing around in the buffer, move them to an NG agar plate with OP50 bacteria using the pulled capillary pipette. Place the plate at 20 degrees Celsius for two to three hours until the worms have recovered and are moving around. Once recovered, individually transfer the worms to NG agar plates with OP50 Then, transfer the plates to a 25 degrees Celsius incubator.
