Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

To study gene expression during xenopus embryonic development, cloned gene products must be stably integrated into the genome. First sperm nuclei are isolated from adult frog testes and perme. Then interphase cytosolic extracts are prepared from xenopus eggs.

The sperm nuclei and egg extract are mixed with linearized transgenic, DNA and a restriction enzyme. During this step, the egg extract decon condenses the sperm chromatin while the restriction enzyme promotes integration of the transgene. Finally, the treated nuclei are transferred into unfertilized eggs.

Each cell of the resulting embryo carries the transgene and promoter dependent expression of the transgene is detected by immunofluorescence microscopy or other methods. The main advantage of this method over other methods, such as DNA injection, is that the resulting embryos undergo integration of the transgene before the first cleavage. This means that the resulting embryos aren't chimeric, and so no breeding is required.

Using this protocol, large numbers of transgenic embryos can be created rapidly and efficiently in only a few hours. These transgenic embryos can be used for analyzing promoter or gene function, or can be bred to create transgenic lines Peek in this procedure by transferring the testes of one to two sacrificed frogs to a dry 60 millimeter Petri dish. Using forceps, macerate the testes until there are no visible chunks.

Add two milliliters of cold nuclear preparation buffer or NPB to the macerate and gently pipette up and down squirt macerate through about four thicknesses of cheesecloth into a funnel, and collect the filtrate in a 15 milliliter tube. Then rinse the dish with eight milliliters of NPB and filter through the cheesecloth into the tube. This solution contains the sperm with gloved hands.

Squeeze the cheesecloth to collect the remaining liquid centrifuge the tube for 10 minutes at four degrees Celsius to pellet the sperm following the centrifugation, decant the supernatant. Then add eight milliliters of NPB and pipe it up and down to resuspend the pellet. Spin down again during the spin, bring one milliliter of NPB up to room temperature.

Then following the spin, resuspend the pellet in one milliliter of NPB and add 50 microliters of 10 milligram per milliliter. Freshly made lyin to perme. The sperm mix gently incubate for five minutes at room temperature.

Next, add 10 milliliters of cold one XMPB with 3%BSA to the tube. To stop the lyan reaction, spin down the lyo than treated. Pellet should look slightly more translucent than it did before.

Resuspend the pellet in five milliliters of cold NPB with 0.3%BSA to wash the sperm nuclei mixed gently with a 10 milliliter pipette and spin down following the spin, decant the supernatant and resuspend the pellet. In 500 microliters of sperm storage buffer transfer the sperm to a 1.5 milliliter tube. This is the nuclei stock Place this tube on ice to check the yield of nuclei.

Place 98 microliters of sperm dilution buffer, one microliter of the nuclear stock and one microliter of a one to 100 dilution of hux stock in a 1.5 milliliter micro fuge tube. Mix well. Using a pipet man allow a small amount to flow into the chamber of a hemo cytometer by capillary action.

Under a compound microscope, count the nuclei. One male should yield 50 to 100 million cells. Place the nuclei at four degrees Celsius overnight to allow the glycerol to penetrate for better cryopreservation.

Liquid nitrogen frozen aliquots are stored at minus 80 degrees Celsius deed eggs collected from eight to 12. Frogs are transferred to a beaker and washed four times in about 35 milliliters of extract buffer, and then two times in 25 milliliters of cytostatic factor Extract buffer or C-S-F-X-B with protease inhibitors to pack the eggs, transfer them into Beckman UltraClear tubes. Allow the eggs to settle.

Remove as much C-S-F-X-B as possible and centrifuge at 150 Gs for about 60 seconds. Following the centrifugation, remove the excess solution from the top of the packed eggs. Then to crush the eggs and generate cytoplasmic extract, centrifuge the eggs at 16, 000 Gs for 10 minutes At four degrees Celsius, the eggs should separate into three layers.

The top layer consists of lipid. The middle layer is cytoplasm, and the bottom layer is yolk. Insert an 18 gauge needle with attached syringe through the tube at the base of the middle layer and withdraw the cytoplasmic solution.

Transfer the solution to a fresh UltraClear Beckman tube on ice. Add protease inhibitors to the isolated cytoplasm to a final concentration of one x centrifuge. The cytoplasmic preparation at 16, 000 Gs for 10 minutes.

At four degrees Celsius, transfer the clarified cytoplasm to a new tube. Expect to obtain 0.75 to one milliliter of cytoplasm per frog. Next, add one 20th of the extract volume of energy mix to the sample.

Transfer the cytoplasm into thick walled polycarbonate tubes. For ultracentrifugation, add one molar calcium chloride to each tube to a final concentration of 0.4. Millimolar incubate the tubes for 15 minutes At room temperature, this inactivates cytostatic factor and pushes the extract into interphase centrifuge.

In an ultracentrifuge at 200, 000 Gs for one and a half hours at four degrees Celsius, the cytoplasm will fractionate into four layers from top to bottom. They are lipid, cytosol, membrane, and mitochondria, and glycogen and ribosomes. Insert a syringe into the top of the tube and remove the cytosolic layer.

This should be about one milliliter. If two to three milliliters was loaded into the tube, transfer the cytosolic fraction to fresh tubes and centrifuge the samples at 200, 000 Gs for 20 minutes at four degrees Celsius, aliquot the supernatant into 25 microliter aliquots in 0.5 milliliter tubes. Quick freeze the aliquots in liquid nitrogen and store them at minus 80 degrees Celsius until use.

To set up a transgenesis reaction, use a clipped pipette tip to gently mix the nuclei stock and combine four microliters of nuclei and two microliters of linearized plasmid in a 1.5 milliliter micro tube. Incubate for five minutes at room temperature while the incubation is proceeding. Dilute 0.5 microliters of the restriction enzyme in 4.5 microliters of water.

Then add one microliter of diluted enzyme to 18 microliters of sperm dilution buffer, two microliters of magnesium chloride and two microliters of high speed extract. Add this mixture to the nuclei plasmid reaction. Incubate 10 more minutes to swell the nuclei during this incubation.

Eggs obtained from two to three female frogs are deed and washed five times in one XMMR. Use a wide boar pipette to transfer 400 to 500 deed eggs to each agros. Well dish containing 0.2 XMMR and 4%fi.

Call approximately 15 to 20 minutes after beginning the transgenesis reaction. Gently mix the nuclei, extract DNA reaction with EC clipped tip and add five microliters of the reaction to 150 microliters of sperm dilution buffer at room temperature to back load the needle. Place a piece of fine TIG on tubing onto the end of EC clipped 200 microliter pipette tip.

Draw the solution in. Then attach the tubing to the needle and allow the solution to enter the needle by gravity. Attach the needle to the pump tubing.

Place the eggs on the microscope stage. Then rapidly insert the needle into the first egg. The injection should be shallow and approximately perpendicular to the X plasma membrane.

To avoid damage, keep the needle in the egg for about 0.5 seconds. Dispensing at a rate of about 20 nanoliters per second, one to two dishes of transplantations can be completed within 20 to 30 minutes. After injections, place the dishes at 16 to 20 degrees Celsius until embryos have reached the four to eight cell stage.

Transgenic embryos resulting from nuclear transfers should be raised until expression from the promoter of interest is visible in the image shown here. A muscle actin promoter drives expression of green fluorescent protein in the somites of this transgenic tadpole. After watching this video, you should have a good understanding of how to use nuclear transfer of sperm nuclei to create transgenic opus embryos.

This approach has been demonstrated for transgenesis with opus lavis, but can also be adapted to zap tropic with minor modifications.