Nuclear Transplantation Reagents and Equipment

1. 1X MMR Prepared as described in Subheading 2.1., item 2.

2. 2.5% Cysteine in 1X MMR (titrate to pH 8.0 with NaOH). Make up fresh each day.

5. 0.4X MMR + 6% (w/v) Ficoll (Sigma Type 400; F-4375) Sterilize by filtration.

6. 0.1X MMR + 50 pg/mL gentamycin (a 10 mg/mL stock solution may be purchased from Gibco-BRL; cat #15710-015). Add 6% (w/v) Ficoll for culturing embryos prior to gastrulation. Culture embryos in 0.1X MMR without Ficoll after gastrulation. Sterilize by filtration.

7. Progesterone (Sigma P-0130; SmM stock in EtOH).

8. Linearized plasmid (200-250 ng/pL): Although we have primarily used plasmid linearized with XbaI or NotI in transplantation reactions, we have also used Xhol, BamHI, and EagI successfully. We think that most enzymes that function in the moderately high salt conditions of the egg extract are likely to work. We commonly purify linearized plasmid for transgenesis using the Geneclean kit by Bio 101, Inc. (cat. no. 1001-200; 1070 Joshua Way, Vista CA 92083; 1-800-4246101). Plasmid DNA can be eluted in either dH20 or in TE (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). If linearized plasmid DNA needs to be concentrated, standard precipitation with 0.1 vol sodium acetate (3 M stock; pH 5.2) and 2.5 vol absolute ethanol, followed by a 70% ethanol wash, can be used. We have found that plas-mid DNA purified in this manner works well for making transgenic embryos and does not adversely effect embryonic development.

We have used enzymes purchased from Boeringer Mannheim or New England Biolabs for transplantation reactions. Some calibration may be required to determine the optimal amount of enzyme to add to each reaction, since additions of 0.5 |L of undiluted enzyme to reactions can adversely affect the development of nuclear transplant embryos. We generally test several dilutions of enzyme (1:2.5, 1:5; 1:10) to identify a dose that has no apparent deleterious effects on transplant embryo development when compared with embryos produced with no enzyme addition.

9. Agarose-coated injection dishes: 2.5% agarose in dH2O is poured into 35-mm or 60-mm Petri dishes. Before the agarose solidifies, a well template (a rectangular square of Dow-Corning Sylgard 184 elastomer) is laid onto it. After the agarose has solidified and the Sylgard templates have been removed, 1X MMR is poured into each dish to prevent dehydration. The dishes are then wrapped in parafilm and stored at 4°C (weeks to months) until use.

10. Transplantation needles: 30-|L Drummond micropipets (Fisher, cat. #: 21-170J) are pulled to produce large needles with long, gently sloping tips (Fig. 2). A micropipet (1 mm wide; 8 cm long) is first heated in a Bunsen burner flame and drawn by hand to make the bore of the needle (200-400 |im wide). This drawn pipet should be 10-15 cm in length and should remain fairly straight when held by one end. To produce a gently sloping needle tip, this pipet is drawn again. We use a gravity-driven needle puller for this: the upper end of the needle is fixed in a brace, the center of the needle bore of the drawn pipet is placed within a small heating coil, and a weight is attached to the lower end of the needle. The gravity driven pullers we have used are home-built and about 10-20 yr old, but similar vertical pullers are commercially available from Narishige (i.e., Model PB-7). The second pull can also be performed with a horizontal needle puller available from Sutter Instrument Co. (Model P-87; Flaming/Brown micropipet puller) using settings like those used to make other injection needles. In limited trials of the Sutter puller using a standard setting, we have found that the needles produced had a steeper slope near the tip and were slightly more difficult to use than those drawn with our vertical puller; however, settings can probably be adjusted on this and other commercially available pullers to produce long, gently sloping tips that will work well for transplantation. Needles are clipped with a forceps to produce a beveled tip of 60-75 |im diameter (see inset in Fig. 2), using the ocular micrometer of a dissecting microscope for measurement.

11. Transplantation apparatus: We have found most commercial injection apparatuses commonly used for RNA and DNA injections unsuitable for nuclear transplantation. This is largely due to the difference in needle tip size. Flow through the 5-10 | m needle tips used for fluid injections can be controlled at fairly high pressures. However, with standard air-injection systems, we have been unable to obtain the extremely low positive pressure, and gentle, controlled flow required to deliver an intact nucleus in a small volume (10-15 nL) through the 50-70 |im tips of nuclear transplantation needles. Oil-filled injection systems (Drummond) are likely to work, since they are based on a positive displacement mechanism that should not be affected by the tip size of the needle. At this writing, though, o

Fig. 2. Diagram of injection apparatus. A pressure regulator is set up on the house air, and a line connecting the regulator is split with a T-shaped connector into an exhaust tube and line to a three-way valve. Another line connects the house vaccuum to the three-way valve. Finally, another line connects the three-way valve to the needle. By adjusting the valves in the three-way valve, the air pressure, and the clamp on the exhaust tube, one can very finely control the level of positive or negative pressure going into the needle. The rectangular inset shows how the needle should appear after the first and second pulls. The circular inset shows how the point of the needle should appear after it is clipped.

Fig. 2. Diagram of injection apparatus. A pressure regulator is set up on the house air, and a line connecting the regulator is split with a T-shaped connector into an exhaust tube and line to a three-way valve. Another line connects the house vaccuum to the three-way valve. Finally, another line connects the three-way valve to the needle. By adjusting the valves in the three-way valve, the air pressure, and the clamp on the exhaust tube, one can very finely control the level of positive or negative pressure going into the needle. The rectangular inset shows how the needle should appear after the first and second pulls. The circular inset shows how the point of the needle should appear after it is clipped.

we have not tried one of these injection apparatuses for nuclear transplantations. Instead, we will describe how to make a home-made air injection apparatus that works extremely well for nuclear transplantions on a large scale and that costs very little (approx $200).

The transplantation apparatus that has given us the most success is shown in Fig. 2. A line connects the house vacuum outlet to a three way valve. Another line connects the house air outlet to a T-connector that splits the air flow into an exhaust line and another line connecting to the three way valve. Finally, another line connects the three way valve to the needle. For fine control of the positive pressure into the needle a screw clamp is placed on the exhaust line. Screwing down on this clamp increases the positive pressure into the system, while opening the clamp decreases the positive pressure. Negative pressure is established by opening slightly the valve (on the three way valve) connected to the house vacuum line. A more rough adjustment of positive pressure also can be obtained by opening or closing the valve (on the three way valve) connected to the house air line. By using a combination of these adjustments, we are able to obtain a very slow, controllable flow through a 50-70-^m needle. As flow is continuous, transplantations can usually be done more rapidly than injections of RNA or DNA, since it is only necessary to move from egg to egg to deliver nuclei. Parts needed to build the transplantation apparatus shown in Fig. 2 are listed in Table 1.

Alternatively, a transplantation apparatus like the one shown in Fig. 3 can be constructed. For this apparatus, a large, air-filled Hamilton Syringe (30 cc Multifit Interchangeable syringe with Luer-Lok tip; Fisher) is connected to a length of Tygon tubing. A metal plunger removed from a Syringe Microburet (Model # SB2; Micro-metric Instrument Co., Cleveland, OH) is used to control injection of the nuclei. We have found this apparatus usable although it is not controlled as easily as the one shown in Fig. 2.

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