Secondary Structure in Pipets

The appropriate bends need to be created in the pipets with the microforge, so they can enter the injection chamber and allow the tips of the pipets to remain at the same focal level when they are being moved in a horizontal plane. The exact angles depend on the type of chamber being used. To allow entry into the injection chamber constructed from a bac teriological dish, both the injection and holding pipets are bent at an angle of around 30°, approx 4 mm from the respective tips (Fig. 1). It is important to orientate the injection pipet correctly on the microforge, so that once the bend has been created, the bevel of the pipet will be in the desired orientation when assembled on the micromanipulation arm.

2.3.2. Micromanipulator Assembly

This section describes the micromanipulatory, optical, and suction systems, and the orientation of the pipets in the injection chamber.

2.3.2.1. Manipulators and Optics

Embryo injections are performed using hand-operated micromanipulators to control the three-dimensional movement of the two pipets in the injection chamber precisely. The two brands commonly used by most researchers are Leitz and Narishige. One micromanipulation arm is mounted on each side of the microscope. The injection and holding pipets can be mounted on either of these micromanipulators depending on comfort for the operator. For embryo injections, ideally an inverted, phase contrast microscope with a magnification range of 40X to 200X and with a fixed stage should be used (e.g., Nikon Diaphot; Leitz Diavert). Such microscopes provide a large working distance between the stage and the condenser to accommodate the injection chamber, and to facilitate simple entry of the pipets into the chamber. Phase-contrast optics are more than satisfactory; however, if Differential Interference Contrast objectives are used, then the injection chamber must be constructed of glass and not plastic. Normal upright microscopes may also be used; however, because of the shorter working distance, a hanging drop injection chamber must be used. This may be constructed from a normal glass microscope slide with two lengths of glass, 22-mm long and 2-mm square, adhered to the longitudinal edges of the slide with Vaseline to support a normal square glass coverslip, also held in place with Vaseline. A small volume of medium is introduced into the middle of the cavity, and each side is back-filled with mineral oil to prevent evaporation.

2.3.2.2. Microsuction

The two instrument holders on the manipulation arms are each connected to micrometer spring-loaded syringes using thick-walled (Portex) tubing (1.0-2.5 mm id). The tubing is filled with lightweight inert min eral oil (Fluorinert 77 or paraffin oil). For ease of operating, each micro-syringe should be placed on the opposite side to the manipulator that controls the movement of the pipet. In this way, both the movement and suction of one pipet can be controlled simultaneously with bothhands. For the holding pipet, a 0.5- to 1.0-mL ground glass syringe may be used. To allow finer control of the suction with the injection pipet, a 50- to 250-|oL Hamilton syringe is ideal. Before fixing the pipets into the respective instrument holders, the tubing must be purged of air bubbles using a 20-mL reservoir syringe filled with inert oil, connected into the hydraulic line via a three-way tap. The pipets are locked into the instrument holders, and using the reservoir syringes, each pipet is filled with inert oil.

2.3.2.3. Orientation within the Injection Chamber

The pipets are aligned in the injection chamber with the tips parallel to the bottom surface, so that they remain in the same focal level when moving in a horizontal plane (Fig. 1). The type of injection chamber used dictates the secondary structure of the pipets and the arrangement of the micromanipulation arms to allow entry into the chamber. The injection chamber recommended here is prepared by simply introducing a 250-|aL drop of medium into the center of a 90mm plastic bacteriological dish, overlayered with lightweight paraffin oil. Thus, a 30° bend created at the tips of the pipets means that with the instrument holders angled at 60°, the tip of the pipets are parallel with the bottom of the chamber. With the hanging drop injection chamber, the pipets must enter the chamber horizontally, and thus, the secondary bends must allow for this different orientation.

Once the pipets are positioned within the chamber, medium from the chamber is drawn a short distance into each pipet using the reservoir syringes, and then the three-way taps are turned back to the micrometer-controlled syringes. Several hundred freshly trypsinized ES cells are then introduced into the chamber and allowed to settle to the bottom before commencing injections.

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