Microinjection of Drosophila Nurse Cells: A Method of Intracellular Delivery
Transcrição
– Place a coverslip with dissected egg chambers onto the microscope stage. Locate an egg chamber of the appropriate developmental stage and orientation for microinjection. Mid para late-stage egg chambers have a large visible oocyte yolk, and those de the correct orientation are with their anterior-posterior axis perpendicular para the direction of injection.
Drosophila nerve cells are a group of 15 cells inside the egg chamber responsible for depositing maternally derived nutrients and biomolecules into the developing oocyte. Set up the microinjector system para the appropriate injection and compensation pressure para ensure consistent injection volume and outward flow. Carefully bring a previously loaded capillary needle into the field of view so that the cells and needle are de the same focal plane.
Ensure that there is no air trapped de the tip of the capillary and that there is a continuous flow of the solution. Then adjust the Z-position of the objective para focus on the membrane separating the follicle cells from the nurse cells, insert the needle into a nurse cell and perform the injection.
The following protocol demonstrates microinjecting Drosophila nurse cells with fluorescent oligonucleotide probes, called molecular beacons, against maternally contributed mRNA transcripts for live cell imaging.
– For molecular beacon microinjection, select the 40x oil objective and mount a cover slip with the dissected egg chamber onto the microscope stage. Locate an egg chamber at the mid para late developmental stage that is properly oriented for microinjection, then set up a microinjector with the appropriate injection and compensation pressures.
Using the micromanipulator joystick, gently lower a needle loaded with one microliter of molecular beacon solution into the oil drop. Bring the tip into focus toward the periphery of the field of view. Perform a clean function para remove the air from the needle tip and para ensure that there is flow from the needle, and bring the needle para the home position.
Focus on the egg chamber para be microinjected, and bring the needle back into focus near the edge of the egg chamber. Perform a fine adjustment of the Z-position of the objective such that the membrane separating the follicle cells from the nurse cells is de focus. Insert the needle into a nurse cell for injection of the molecular beacons over a period of two para five seconds.
– To ensure reproducible microinjection success, focus carefully on the membrane separating the follicle cells and the nurse cells that will be injected, while bringing the tip of the needle into focus with the micromanipulator.
– When all of the molecular beacons have been injected, gently remove the needle and retract it para the home position. Change the objective para the desired magnification for image acquisition, then focus on the egg chamber under the new objective and begin the image acquisition.
