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Xenopus embryo injections: Inducing Ovulation of female Xenopus: 1) Get frog the night before (check which tank has available frogs; fill in the sign out sheet); inject each female frog with HCG (800 units/frog). Check whether you will need to sacrifice a male to get testis; if so, bring one up. Generally, place female frogs ON in 16 degree incubator (usually about 14 hrs. at 16ºC should be sufficient to induce egg laying) Next day: 2) As soon as possible in the morning, take frogs out of incubator, place at RT. Replace frog water with RT frog water from large carboy. If frog is ready, induce the frog to lay eggs. Often, you can tell if frog is ready to lay by bright red cloaca, or the presence of eggs in the water already (“dumping”). If frog is not laying eggs, let it sit at RT for a while and try to induce laying again at 45-60 minute intervals. 3) Fertilization of eggs: --ovulate eggs from female directly into 1x MMR --Look at eggs under microscope: pock marks? pigment of animal hemisphere ok? other? If ok, proceed. If not, get different eggs. --Drain MMR from the dish --Get testes/sperm. Place one testis in the dish with the eggs and tear off a piece with a forcep (usually 1/10-1/20th of one testis or less per fertilization is sufficient if it is fresh; check date on vial; when testis is >1 wk old use more for each fertilization and discard testis over 2 wks. old) --use forceps to macerate the testis, breaking open each of the small compartments that contain the sperm. --gently swirl and rock the dish briefly to distribute the sperm macerate over all of the eggs --Add 0.2x MMR to just cover the eggs. Break open any intact pieces of testis that are still visible in the dish by crushing or tearing with a forcep --Watch for “activation” of eggs: -contraction of pigmented animal hemisphere (pigment appears to recede to make “beanie cap eggs”) followed by relaxation back to near original appearance (will occur 5-10 minutes after fertilization . -all eggs turn dark side up. -wait 10-15 minutes, until all are activated. --In the meantime, make cysteine solution: 1.25 g of cysteine in 50 ml of water, + about 6 drops of NaOH (pH soln with paper until it has a pH of between 7.8-8.5) --Drain 0.2x MMR off of eggs, add cysteine solution. --wait 3-4 minutes as jelly coat comes off of eggs. --eggs clump together at bottom, “ghosts” come off. --4-5 washes with 0.2x MMR (after final wash: place eggs in 0.2x MMR+ 4% ficol). --aliquot eggs into 3 60 mm dishes and place one in each incubator (14, 16, 18ºC) --incubate 1.5-2 hours (at diff’t temps if you want the cleavage of different groups of eggs to be spaced out) until cleavage is underway. 4) The injection of the eggs: Injector: -power on -air on -air pressure entering system is visible with knob turned to setting: “P clear” -turn setting to “P inject” --pressure should read >= 2.8 --adjust with knob (P inject) -inject time: 100-600 msec---calibrate to your needle. Droplet calibration of the needle: You want each injection to be a release of 10 nL from the needle. 10 nL is about 14 small units of the eyepiece reticle (ruler) at the highest magnification (50X). -hook needle to tubing (about 1.5 cm in the tubing) -clamp needle/tubing gently into the electrode holder. -turn to lowest magnification so you can manipulate needle into dish. -center tip of needle in viewing field approximately so that when it is in the focal plane used for injections you can see the tip in focus -fill needle with H2O (using the fill pedal). -inject a few droplets into oil (the first drop is not always representative), measure size of droplet, adjust injection pressure and/or inject time in order to make the drop size 14 units. -press “clear” to eject water. -Now, place a 1-2 µl drop of your plasmid or RNA onto the RNase free (clean) side of a small square of parafilm and draw it into the needle using the vacuum foot pedal. Don't get too greedy as if you suck up air you will lose everything previously loaded into the needle --place eggs to be injected into a 60 mm dish of 0.2XMMR + 4% Ficoll +100µg/ml gentamycin. Swirl eggs to make a pile in the center. Use one blunt (size 3) forcep to nudge your first egg toward the needle tip. Then use both forcep tips as a firm support behind the egg to keep it from slipping away as you bring the needle in and inject with the other hand controlling the needle through the micromanipulator 5) When done, clear your needle. Turn off air, allow pressure to go down all the way. Once pressure is down to zero, you can turn the power off. Testis isolation: 1) bring up a male frog (generally the darker and stickier his mating pads are the better the testis size and quality will be)--lower water in the bucket to 1 liter and add 5 mls of benzocaine stock (10% in EtOH: stored at 4ºC) 2) Wait about 20 minutes until he has been lethally over-anaesthetized 3) make a slit through the ventral skin and body wall muscle and find the testis behind, attached to and at the base of the large yellow fat bodies. Cut these off, being sure not to nick or tear them. 4) If necessary, cut attached excess fat away from the testis (a little is okay as one can use it to handle the testis); then place the two testes in a clean glass vial of sterile 1XMMR + 100 µg/ml gentamycin. Store testis on ice or at 4ºC whenever they are in use to prolong life. 5) After isolating the testis, cut the heart out of the male or pith as an assurance of death. Wrap the body in a paper towel or glove and place in the freezer morgue (-20ºC bottom shelf) Cutting animal cap explants: --Get your fertilization going early in the day (by 9-11 AM) --Do your injections and transfer injected eggs to 6 well plates in 1XMMR +4% Ficoll+ 100 µg/ml gentamycin (Try to finish by 1-2 PM; no later than 3PM unless you are not adverse to staying late). Generally, you will need about 10 animal caps for each sample type; I would recommend injecting about 15 embryos to account for 1-2 not making useable blastula and 1-2 of your caps falling apart or dying. This may not be necessary if egg quality is high. Remember to save some uninjected embryos to generate both uninjected explants (negative control) and whole embryo RNA at the same stage (positive control). These embryos need to be kept at the same temperatures and treated in the same manner as your injected samples. --Place developing embryos in a 25ºC incubator or somewhere in the main lab that is equally warm to generate blastula stage embryos rapidly. --About 3-5 hours after finishing injections (depending on how extensive the experiment is and how long you were injecting) the first injected embryos will reach stage 8 (see Nieuwkoop and Faber images on wall) --You will cut caps in 1XMMR (filter-sterile; make up a 500 ml bottle). Transfer each group of injected embryos to a fresh 60 mm dish filled with this solution. Remove the vitelline envelope with a relatively sharp pair of forceps (remember that you can pierce the egg with forceps in the marginal or vegetal zone without damaging cells that will end up in your explant). Place the embryo animal side up, being certain that it isn't tilted sideways. I generally remove vitelline envelopes from all of the embryos before cutting the caps --Use your forceps to pinch off the explant. The cap should be a square (shape very approximate) of tissue from center (top) of the animal hemisphere. I usually visually divide the animal hemisphere into thirds each way and try to cut my square from the center third. --After cutting, caps should be transferred using a P200 pipetteman and cut-off pipette tip to wells of a 12-well plate containing (0.7XMMR + 1mg/ml BSA +100µg/ml gentamycin; sterile). I make this solution up fresh in a 50 ml conical for each experiment using my sterile 1XMMR stock (35 mls 1XMMR, 15 mls sterile H2O, 50 mg BSA solid). --Culture explants at 16-18ºC until they reach the desired stage for analyzing gene expression. To stage explants requires that you carry intact embryos through every stage of this manipulation at the same temperature that the within 30-60 minutes, if not sooner, the explant should round up into a tight ball. Unless you have contaminated this ectodermal explant with mesoderm, explants of uninjected cells should still be round (no extension or elongation of the tissue piece) the next day.
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