Gonad Microinjection: A Method of Compound Delivery Directly into the Germline of C. elegans
Transcrição
– To prepare for the injection, use a worm pick para lay a track of halocarbon oil on an agarose pad. Place worms de the oil and immobilize them by gently pushing them onto the agar pad. Ensure the oil fully covers the animals. It allows oxygen para get para the worms while preventing desiccation. Next, place the agarose pad on an injection microscope and position the needle perpendicular para 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 para an NGM agar plate with bacterial food. In the following protocol, the gonad microinjection method is used para deliver a Cas9 complex para target and edit the dpy-10 gene.
– Begin with preparation of C. elegans and agarose pads as described de the text protocol. Place an agarose injection pad and cover slip onto a dissection scope. Use a worm pick para lay a small track of halocarbon oil along one edge of the pad. Then, use the worm pick coated de oil para lift several worms off the NG agar plate and into the track of oil. With a fine hair attached para a pipette, such as an eyelash or cat whisker, position the worms de parallel gently pushing the worms into the agarose pad. Até comfortable with the microinjection procedure, only mount and inject one worm at a time.
Once de position and attached para 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 para the injection needle, which has been filled with the RNP solution, as described de the text protocol.
Switch para a high magnification and reposition the needle adjacent para the gonad arm, corresponding para the region near the nuclei de mid- para 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 para jolt the needle through the cuticle. Depress the injection pedal or button, slowly fill para 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 para release the worms from the agar. After 10 minutes, when the worms are thrashing around de the buffer, move them para an NG agar plate with OP50 bacteria using the pulled capillary pipette. Place the plate at 20 degrees Celsius for two para three hours until the worms have recovered and are moving around. Once recovered, individually transfer the worms para NG agar plates with OP50 Then, transfer the plates para a 25 degrees Celsius incubator.
