Materials

1. Inverted microscope (e.g., Nikon Diaphot, Tokyo, Japan) with the following features:

a. Image-erected optics.

b. A condenser with a long working distance.

c. A fixed stage (the objective lens rather than the stage moves when focusing).

d. 10x Magnification eyepieces.

e. 4x Objective for low-magnification work.

f. 40x Objective for microinjection.

g. Suitable objectives: Nomarski differential interference contrast (DIC) optics are best for visualizing the internal structures of the egg. DIC optics are expensive, and glass-injection systems are required. Hoffman modulation contrast optics are compatible with plastic injection chambers, but give inferior resolution to DIC. If both are not available, eggs can be viewed under bright field. Phase-contrast microscopy is not compatible with any microinjection system.

The Nikon Diaphot-TMD equipped with the diascopic DIC Normarski attachment TMD-NT2 is excellent, robust, affordable, and widely used for microinjection.

2. Micromanipulators: Two are required—for the holding pipet, which holds the egg in place, and for the microinjection pipet. The Leitz M (Wetzlor, Germany) type, with joystick control of horizontal movement in two planes, is most commonly used. The micromanipulators and the microscope must be positioned on a purpose-built baseplate (Fig. 5), which must be custom-engineered. Narishige produces economically priced micromanipulation systems compatible with its Diaphot-TMD microscope. These are very flexible and do not require a custom-built base plate.

3. Micropipet holders: Leitz single-instrument holders fitted with Leitz singleinstrument tubes if Leitz Type M micromanipulators are used.

a. Draw a Leitz hard-glass capillary (cat. no. 520119) in a gas flame. Grip the ends of the capillary, and turn the middle of the capillary in the hottest part of the flame until the glass softens. Quickly withdraw the pipet from the flame, and simultaneously pull on both ends.

b. Score the drawn section 2 cm from the shoulder with a diamond pen, and break the capillary at this point.

c. Mount the capillary vertically in a microforge (e.g., Narishige MF-9). Focus on the tip of the capillary with the 4x objective. The capillary must be straight and its tip perfectly flush with no jagged edges and an external diameter of 100 mm (±20 mm). Bring the tip close to, but not touching the microforge filament. Heat the filament, and melt the tip until its internal diameter reaches 10-15 mm. The size of the hole is extremely important; too small a hole will make controlling the holding pipet difficult; too large a hole may allow eggs to be sucked into the pipet.

d. 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 will last one microinjection session and is not reused. Large numbers can be made in advance and stored in a sterile plastic tube.

5. Microinjection pipets for physically introducing the DNA fragment into the nucleus of the one-celled egg: Microinjection pipets are made from thin-walled glass capillaries with an external diameter of 1 mm. Capillaries with an internal filament (Clark Electromedical Instruments, Reading, UK; cat. no. GC 100 TF-15) are useful, since they can be backfilled by capillary action from the distal end to the injection tip. Pipets are drawn on a commercially available pipet puller (e.g., David Kopf Instruments, Tujunga, CA; Vertical Pipet Puller Model 720 or the Narishige PB-7). Injection tips should have a 1-mm opening. Larger tips burst eggs, and smaller ones become blocked easily. Microinjection pipets are pulled when needed.

6. The injection chamber: Glass depression slides are compatible with inverted microscopes fitted with any optic system. Siliconize slides by rinsing in a 3% (v/v) solution of dichloromethyl silane in chloroform. The slides are rinsed thoroughly with water and a standard household detergent. Prior to use, rinse the slide in ethanol, and dry with a paper tissue. It must be devoid of dust particles.

7. Agla micrometer syringe (Wellcome, Kent, UK, or equivalent).

Fig. 5. A typical arrangement of the equipment needed for the microinjection of fertilized one-cell eggs. A. Agla micrometer syringe. 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).

Fig. 5. A typical arrangement of the equipment needed for the microinjection of fertilized one-cell eggs. A. Agla micrometer syringe. 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).

Microinjection Holding Pipette Size
Fig. 6. Construction of a typical holding pipet.

8. There are two possible microinjection systems:

a. Manual injections involve the use of a 50-mL syringe with a ground-glass plunger connected by an air-filled tube to the microinjection needle. Manual squeezing of the syringe squeezes DNA from the pipet into the egg.

b. An automatic injection system that uses compressed air to expel the DNA, triggered by a foot-operated pedal (e.g., Narishige: Picoinjector PL1-188 with compressed air supplied from the Hitachi Package Oilfree Bebicon PO-O 75PSB compressor). Although more expensive than manual systems, the advantages are:

i. The foot-operated injection trigger leaves both hands free to control the micromanipulators.

ii. A constant low positive pressure applied to the microinjection pipet produces a continual outflow of the DNA, preventing the backflow of M2 medium and clogging of the pipets.

The assembly of an automatic injection system should be done by a trained professional. The injection pressure must be determined empirically.

9. M2 and M16 culture media.

11. Light liquid paraffin (Fluka 76235).

12. 26-Gage needles.

13. Tygon (Akron, OH) tubing (3/32-in. id; 5/32-in. od).

14. One clamp stand.

15. Diamond pen.

16. Disposable 1-mL syringes.

17. Fluorinert electronic liquid (3M Company, St. Paul, MN; cat. no. FC77).

18. Dissecting microscope.

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