Xenbase: a Xenopus web resource

Genetics: transgenics

4. Preparation of DNA, Needles and Equipment (or go back to 3. prepare nuclei)

A. Preparation of linearised DNA.

Digest DNA using standard conditions, run the DNA on a 1XTAE agarose gel, isolate the appropriate band and purify using the Geneclean Kit II by Bio101. Elute the DNA in dH2 O at a final concentration of 150-250 µg/ml.

B. Preparation of nuclear transplantation needles.

These needles are unlike standard needles used for DNA and RNA injection in that the diameter of the point is an order of magnitude larger to allow nuclei to pass through intact. 30 µl Drummond micropipets (Fisher, cat. #: 21-170J) are used to make the needles. It is important to pull needles with gentle slopes at the tip. This makes it easier to clip the needles at the desired diameter and also they cause less damage to the eggs during transplantation.

We currently use a Flaming/Brown Micropipette Puller Model P-87 (Sutter Instruments Co.) for pulling our needles. The setting depend on the filament used, so should be adjusted so that a gentle slope toward the tip is achieved. Clip the needle with forceps to produce a bevelled tip of 80-100 um diameter using the ocular micrometer of a dissecting microscope or a stage micrometer for measurement. It is essential that the tip be this wide or nuclei passing through will be damaged. When clipping tips, it often helps to use forceps with slightly unmatched tips and to pull outward at a 20 or 30 degree angle from the needle as the forceps contacts the needle. Treat the inside of needles with Sigmacote (Sigma SL-2) to prevent shearing of sperm nuclei flowing through the needle (needles can be coated 10 minutes to several months before use).

Attach approximately 1 cm Tygon tubing (R-3603 1/32Ó; Fisher, cat. #: 14-169-1A) to the end of a plastic pipetteman (200µl) tip anduse the pipetteman to draw up Sigmacote; then attach the other end of the tubing to the injection needle (see below). Depress the pipetteman plunger to force Sigmacote through the needle until a few drops emerge from the tip then release the pipetteman plunger to withdraw most of the solution. Rinse needle with at least 200ml of water. Make sure to remove all the liquid from the needle as any remaining liquid will block flow of nuclei into the needle.

C) Agarose-coated injection dishes

Pour 2.5% agarose in 0.1X MMR into around 10 60mm petri dishes. Before the agarose solidifies, place small weigh boats on the agarose so that as the agarose solidifies, a square depression in the agarose remains. The depression will accommodate ~500 eggs. Wrap the dishes in parafilm and store at 4°C until use.

D) Transplantation Apparatus Most commercial injection apparatuses used for DNA and RNA injections which are based on air pressure are unsuitable for nuclear transplantations, due to the large difference in needle tip size. Flow through the 5 µm needle tips used for fluid injections is controllable at fairly high pressures. However, with these standard systems it is usually not possible to obtain an extremely low positive pressure and gentle, controlled flow required to deliver an intact nucleus in a small volume (10-15 nl) through a 80-100 µm needle tip. Therefore, for nuclear tranplantation we use a Harvard Apparatus infusion syringe pump (Model 22; Cat.# 55-2222) with two 2.5ml Hamilton Gas Tight Syringes and Tygon R3603 tubing (0.8mm X 0.8mm). This way two people can transplant nuclei at the same time. The oil that we use to fill the system is Mineral Oil (Embryo Tested) from Sigma (Cat. No. M-8410). The big advantage of using an infusion pump is that we can adjust it to any desirable flowrate. We have ours set at 10nl/sec.

NEXT: 4. make transgenics

contact the page administrator (E.Amaya)