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Genetics: transgenics

4. Transgenesis by sperm nuclear transplantation into unfertilised eggs

1. The night before eggs are needed for transplantations, inject 2 adult female frogs in the dorsal lymph sac with 500-800U Human Chorionic Gonadotropin (HCG) and incubate at 19¡C for 10-12 hours.
2. Remove a 1ml aliquot of Sperm Dilution Buffer (SDB) from the freezer and allow it to warm to room temperature
3. Make up 500ml of 2.5% cysteine in 1X MMR pH 7.8 (with 5N NaOH)
4. Fill agarose coated injection dishes with 0.4X MMR + 6% Ficoll
5. Set up a reaction: 4 µl sperm stock (~4 X 10 5 nuclei) 2.5 µl linearized plasmid (100 ng/µl) Incubate 5min at room temperature. Meanwhile: Obtain a 15ml aliquot of high speed extract from -80 freezer and allow to thaw to room temperature (only takes a few minutes). Keep aliquot on ice for the day.
6. Make the following mixture: 18 µl SDB 2 µl extracts 2 µl 100 mM MgCl2 (add to 5 mM final at all steps to aid enzyme action) 0.5 µl of a ~1:10 dilution of Sal I or Not I to the extracts (depending on the construct). Mix well.
7. After 5 minute incubation of DNA with sperm nuclei add the extract:enzyme:MgCl2 mixture to the sperm nuclei. Mix the reaction by gentle pipetting (using a clipped yellow tip). Incubate 15 min. at room temperature
8. While sperm are swelling in reaction mixture, collect eggs from the frogs and dejelly them in 2.5% cysteine hydrochloride in 1X MMR (pH 8.0 with NaOH). Wash the eggs thoroughly in 1X MMR. Transfer dejellied eggs into agarose coated dishes containing 0.4X MMR with 6% ficoll and gentamycin. We try to fill the depression square with eggs. After about 5 minutes in 0.4X MMR + 6% Ficoll the eggs will pierce easily
9. After the 10-15 minute incubation with extracts, mix the sperm nuclei gently by pipetting up and down with a cut of yellow tip. Then transfer 5 ml of mixture into 150 ml of SDB that is already at room temperature. At this point do not mix the nuclei. Mixing at this point is likely to shear the nuclei. Allow the sperm nuclei:extract mixture to slowing equilibrate with the SDB over the span of a few minutes
10. Using a piece of Tygon tubing attached to a yellow tip (as previously described for Sigmacoting needles) draw up the dilute sperm suspension, mix gently by pipetting up and down and then draw up the dilute sperm nuclei and detach the yellow tip from the pippetman (try not to create or leave bubbles in the tygon tubing as these may damage nuclei). Be careful to keep the yellow tip horizontal or the nuclei will dribble out. Now back load a needle by attaching it to the tygon tubing and raising the angle slightly so that the nuclei flow gently into the needle. As long as no liquid is present at the tip of the needle the nuclei should flow easily by simple gravity. Once the needle has backfilled completely with nuclei,detach the needle and attach it to the tygon tubing filled with mineral oil that is connected to the Harvard Apparatus infusion pump. If two people of injecting load another needle as before. Place the yellow tip with the remaining nuclei aside horizontally in case you need to load another needle.
11. Transplant sperm nuclei into unfertilised eggs. Start the flow in the infusion pump and begin injecting eggs, keeping the needle inside each egg for approximately one second. Move the needle fairly rapidly from egg to egg, piercing the plasma membrane of each egg with a single, sharp motion then drawing the needle out more slowly. The angle of the needle should be perpendicular to the membrane surface (rather than glancing) to avoid tearing the plasma membrane. If the needle becomes clogged with cytoplasm, bring the tip to the air-liquid interface of the dish. Sometimes the surface tension of the interface removes the cytoplasm plug in the end of the needle. If a needle tip is too narrow, or if it becomes partially clogged with debris during transplantations, the injected nuclei will be damaged during transplantation, resulting in aneuploid or haploid embryos. You can determine whether your sperm dilution and the flow rate used for injections were appropriate by watching the first cleavage of the transplanted eggs. If few of the eggs received a nucleus, the frequency of cleavage will be low; one fifth to one third of our transplantations typically result in normally cleaving embryos. Eggs that were injected with more than one nucleus will divide at the time of first cleavage abnormally into three or four (or more) cells. Many of these embryos will develop to blastula stages, but most fail during gastrulation; in some, a region of the embryo will fail to cellularize and die. Eggs injected with multiple nuclei which do gastrulate usually do so abnormally; typically, blastopore closure is incomplete resulting in embryos that form two wings of somites and neural tissue on each side of the exposed yolky tissue lying in the center of the trunk. This type of gastrulation failure is common to stressed or unhealthy embryos (particularly embryos derived from ÔsoftÕ eggs).
12. When the embryos have reached the 4-cell stage, gently separate them from uncleaved eggs and transfer them to a separate dish of 0.1X MMR + 6%Ficoll +50µg/ml gentamycin. Do not be fooled by speudocleaveages. Only keep embryos that appear like normal, healthy 4-cell embryos. We commonly culture transplanted embryos in 24-well tissue culture dishes with about 5 embryos per well, since culturing embryos at high density can compromise their health. It is also important to remove dying embryos promptly since they also can compromise the health of their siblings. When embryos are around stage 12, media is replaced with 0.1X MMR + 50µg/ml gentamycin without Ficoll. Because of the large needle tip used for transplantations, embryos often develop large blebs at the site of injection. These blebs occur when cells are forced out of the hole left in the vitelline membrane at the injection site but they generally do not affect development. The blebs usually fall at the neurula or tailbud stages, but they can be removed manually once the embryos have reached the late blastula stage.

References:

Kroll, K.K. and Amaya, E. (1996) Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation. Development 122:3173-3183.

Murray, A.W. (1991). Cell cycle extracts. In Methods in Cell Biology, (B. K. Kay, and H. B. Peng), ed., Vol. 36, pp. 581-605. San Diego: Academic Press, Inc.

Amaya E, Kroll KL . A method for generating transgenic frog embryos. Methods Mol Biol. 1999;97:393-414.

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