Holding pipets

Place the tip of the holding pipet next to an egg and immobilize it against the tip by applying negative pressure. Attempt to orient the egg so that the pronucleus is... 

Fig. 1. Tools used for micromanipulation of 6.5-8.5-d mouse embryos. A The glass needle, B the holding pipet, C the injection pipet used for delivering dye or DNA solution, D the pipet used for grafting cell clumps, and E the two bends produced in the pipet (shown in B-D) to enable the tip of the pipet to reach over the wall and access the medium drops sitting on the bottom of the culture dish during micromanipulation (for setting up, see Fig. 5B).

The holding pipets are made using Leitz thick-wall glass capillaries. The internal diameter of holding pipets should be 10-50 pm, depending on the size of the embryo used for the experiment. 

Bring the holding pipet into the field of view by pushing it through the oil into the drop. Draw a small amount of medium into the holding pipet to create an oil medium meniscus. 

Release the embryo from the holding pipet by applying a positive pressure via the micrometer syringe. Return the embryo to the culture medium (section 3.1). 

Fig. 7. Labeling of the epiblast by microinjecting Dil into the distal cap of the embryo. To inject in the midline, the embryo is held at the distal tip of the egg cylinder hy gentle suction with the holding pipet (h). The injection pipet (ip) is passed through the extraembryonic tissues of the egg cylinder. The injection pipet is brought to the site of labeling from within the pro-amniotic cavity to avoid inadvertent labeling of other embryonic germ layers. The arrow points to the tip of the labeling pipet in the epiblast layer en, primitive endoderm primitive streak. Bar = 20 pm.

Set up the miCTomanipulation apparatus and attach the holding pipet as described in section 33, steps 6-9, except that an injection pipet and Ught paraffin oil is not used. 

Bring the electrodes into the field of view around the embryo Position the embryo between the two electrodes using the holding pipet as in section 3.3.2, steps 14-16, except that the embryo is held on the side opposite the site of electroporation (Note 19). 

Fig. 8. Labeling the endoderm of 7.0-d embryo by electroporation. A The embryo is first bathed in plasmid DNA solution before transfer to the electroporation drop on the culture dish. To electroporate the distal tip, the embryo is held at the extraembryonic ectoderm by suction using the holding pipet (hp) and positioned between the platinum plate (cathode) and tungsten needle (anode). The electrodes are connected to an Electro Square Porator and the electroporation is focused at the tissue closest to the needle tip. B Fluorescent cells in the distal region of the embryo following electroporation with a P-actin-eGFP construct and cultured for 3h.

Fig. 9. Grafting of cells to a 6.5-d embryo. The embryo is held on the anterior side opposite the intended site of grafting by the holding pipet (h). The cells are grafted into the epiblast (ep) on the posterior side of the embryo ( marks the position of the primitive streak) immediately proximal to the margin of the distal cap. Bar = 20 pm.

Hold the embryo by the yolk sac with the holding pipet by applying negative pressure to the micrometer syringe. The embryo is positioned so that a clear silhouette of the cranial nenral fold is visible. 

The successful culturing of 5.5-d embryos was recently described using similar culture medium. Using a micromanipulator, hold the 5.5-d embryo with a holding pipet and remove the Reichardt membrane a glass needle. Culture in a medium of DMEM RS (1 1 and 1 3 by volume) for up to 48h (11). 

When moving between the dye drop and the media drop, the holding pipet should be retracted, so that its tip does not become contaminated with the dye. 

Micromanipulators Two are required—for the holding pipet, which holds the egg in place, and for the microinjection pipet. The excellent Leica mechanical micromanipulator (Wetzlor, Germany), with joystick control of the horizontal movement in two planes, is most commonly used. The micromanipulators and the microscope must be positioned on a purpose-built base plate (Fig. 5), which must be custom engineered. Narishige (Tokyo, Japan) produces economically priced micromanipulation systems compatible with Nikon microscopes. These are very flexible and do not require a custom-built base plate. 

Fig. 5. A typical arrangement of the equipment needed for the microinjection of fertilized one-cell eggs. A Agla micrometer S5ringe. B Liquid-paraffin-filled tube. C Left-hand micromanipulator. D Inverted microscope. E Base plate. F Camera (optional). G Left-hand instrument tube for holding pipet. H Microinjection chamber (depression slide) sitting on fixed stage. I Right-hand instrument tube for injection pipet. J Air-filled tube. K Glass 50-mL syringe. L Right-hand micromanipulator. M Video system (optional).

Move the capillary to a horizontal position. Position the filament 1-2 mm below the capillary tip. Move the filament close to the pipet, and heat to bend the capillary by about 15° to the horizontal. One holding pipet lasts one microinjection session and is not reused. Large numbers can be made in advance and stored in a sterile plastic tube. 

Clamp the instrument tube into the instrument tube holder of the left-hand micromanipulator (if one is right handed). Adjust the micrometer syringe until fluid stops flowing out of the holding pipet. Do not allow air to flow back into the holding pipet. 

Using the micrometer syringe, draw M2 medium into the holding pipet until the meniscus between the M2 and Huorinert is just at the shoulder of the holding pipet. 

Position the microinjection pipet until the tip is 2cm above the center of the microinjection chamber and 15-20° to the horizontal. Using the fine vertical control of the micromanipulator, lower the tip into the injection chamber until it is just above the chamber floor. Monitor the position of the tip throughout using the 4x objective. Using the horizontal controls of the nuCTomanipulators, bring both the injection pipet and the holding pipet to the center of the field of view. 

Switch to the 40x Nonnarski objective, and focus on the holding pipeL Using the line controls of the micromanipulator, bring the tip of the microinjection pipet into the same focal plane as the holding pipet. The microinjection pipet should move freely in the horizontal plane when operated by the joystick control. 

About 2 cm further away from the tapered shaft, break the tubing with a diamond pen and fire-polish the end to yield an S-shaped holding pipet. When used, the pipette shaft will extend, parallel to the floor of the culture dish, into the microdrop for manipulation (Gordon and Ruddle, 1983). The tubing will then extend vertically up and over the edge of the tissue culture dish, and attach to the instrument collar of the manipulator. 

Using the holding pipet, pick up a blastocyst at the equatorial plane on the side of the inner cell mass. 

Figure 9.7 Wet chuck and micropipet assembly for holding pipet. The chuck is inserted and locked into the nose of the micronianipulator. (Courtesy of Research Instruments Inc., Durham, NC).

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