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[15] Maintenance of Xenopus laevis and Oocyte Injection By ALANL. GOLDIN Introduction
Xenopus laevis, the South African dawed frog, is a member of the anuran family Pipidae. There are six species of Xenopus, all indigenous to Africa, but Xenopus laevis is the only one commonly used in laboratory research.~ Xenopus laevis frogs are distinguished by the presence of three toes with claws on each hind foot of the animal, hence the name, which means "clawed frog." There are four subspecies ofXenopus laevis, termed X. laevis laevis, X. laevis petersi, X. laevis victorianus, and X. laevis borealis. 1 All Xenopus are aerobic but entirely aquatic, so there is an absolute requirement for breathing air but no necessity for any land-based existence. Xenopus are very susceptible to desiccation, and they can die from being out of the water for a few hours. Xenopus frogs are normally darkly pigmented on the dorsal side, but their skin contains chromatophores which rapidly change color according to the surrounding environment. The frog secretes both a mucouslike substance and a toxic substance with properties like epinephrine. These are most likely defense mechanisms for the frog, but they are not harmful to humans when handling the frogs. Xenopus laevis was first used experimentally in a clinical setting as a pregnancy test. Female frogs were injected with urine from a possibly pregnant woman, which resulted in egg laying only if the urine contained human chorionic gonadotropin (hCG), an indication of pregnancy. Although the frogs are no longer used for this purpose, they are still widely used as an experimental system in developmental biology.2 However, the most common use today of Xenopus laevis has been for their oocytes. Gurdon et aL originally demonstrated that Xenopus oocytes can be used to express exogenous mRNA species when microinjected into the cytoplasm) Many investigators have sinced used this property to study the expression and translation of a large number of molecules (for reviews, see Rcfs. 4-8). J. B. Gurdon and H. R. Woodland, in "Handbookof Genetics"(R. C. King, ed.), p. 35. Plenum, New York, 1975. 2I. B. Dawidand T. D. Sargent,Science240, 1443(1988). 3j. B. Gurdon, C. D. Lane, H. R. Woodlandand G. Marbaix, Nature (London) 233, 177 (1971). 4j. B. Gurdonand M. P. Wickens,this series, Vol. 101,p. 370. s A. Colman,in "Transcriptionand Translation--A PracticalApproach"(B. D. Hamesand S. J. Higgins,(Ms.),p. 271. IRL Press, Oxford, 1984. METHODS IN ENZYMOL(X}Y, VOL. 207
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reservod.
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Sources of Xenopus Frogs Although Xenopus laevis is indigenous only to South Africa, the frogs are now available from a number of different suppliers throughout the world (see Appendix II for a listing). There are two major types of frog suppliers, those that capture and sell frogs reared in the wild and those that sell frogs bred in their own facilities. Frogs bred in the laboratory are generally more consistent and can be fed a synthetic diet available from various suppliers (see below). On the other hand, frogs captured in the wild may be less susceptible to disease than inbred frogs .1 They will not eat a synthetic diet such as frog brittle, however, and in fact may die of starvation if kept on such a diet. They must be fed a natural diet consisting of liver, heart, or similar material (see below). Both laboratoryreared and wild-reared frogs can be used successfully for the production of oocytes. The specifics of ordering frogs for oocyte use vary with each supplier, but it is generally a good idea to request the largest and most mature females for the purpose of obtaining stage V oocytes. These large frogs also contain a significant number of less mature oocytes, so that it is possible to obtain oocytes of all stages from them. In addition, it is advisable to request shipping with cold packs during the summer months to prevent extremes of temperature variation. To avoid this problem it may be helpful to have sufficient frogs shipped prior to the hot months of summer. Once the frogs have arrived it is a good idea to let them acclimatize for about 2 weeks before use. In addition, because of the widespread use of Xenopus frogs for oocyte injection experiments, there have been recent shortages, particularly from the suppliers that sell laboratory-bred frogs. To synchronize oocyte development, the frogs can be injected with hCG, which induces ovulation and egg laying, after which oocyte development starts over approximately synchronously. An appropriate dose is 400-500 IU, which should induce egg-laying in about 12 hr? Many suppliers offer flogs which have been injected with hCG. If hCG-injected frogs are to be ordered, however, be sure to request that the frogs be kept for 2 - 3 months after injection to allow sufficient time for mature oocytes to develop. Uninjected frogs generally are just as good a source of mature oocytes as injected ones, so that hCG injection is not usually necessary.
6 H. Soreq, Crit. Rev. Biochem. 18, 199 (1985). 7 T. P. Snutch, Trends Neurosci. 11, 250 (1988). 8 N. Dascal, Crit. Rev. Biochem. 22, 317 (1987).
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M a i n t e n a n c e of Frogs
Environmentfor Frogs Xenopus can tolerate a wide range of temperature fluctuations, but the quality of oocytes diminishes markedly following temperature shifts. The frogs should therefore be housed in a temperature-controlled environment at approximately 20 °. Sufficient air conditioning is especially important, as heat seems to be more detrimental than cold. In addition, the frogs should be maintained in a constant light-dark cycle of 12 hr each. This helps to prevent the seasonal variability found with Xenopus in the wild, although oocyte quality generally does diminish during the summer months. A stable environment will help to ensure that the oocytes are still usable, however. The containers for the frogs need not be particularly sophisticated. Large polypropylene tanks or aquarium tanks both work well. The advantage of polypropylene is that it keeps the frogs more isolated from the external environment, but aquarium glass can be painted black to achieve the same effect. This is beneficial since the frogs are easily startled by movement or light. It is important to have a tight-fitting lid with holes for air, since the frogs can easily lift a lid and escape. Frogs are obligate air breathers, and must be able to reach the surface, so the level of water should not be greater than about 6 - 1 0 inches. Suitable containers are Nalgene tanks 18 by 12 by 12 inches high (Fisher, Pittsburgh, PA, Cat. No. 14-831-330), with holes drilled in the lids. Each of these containers can hold 6 - 8 frogs, with 2 - 3 liters of water per frog. Treatment of Water Xenopus are freshwater frogs that prefer stationary water. If running water is used to keep the water clean, it should be provided at a slow rate to prevent development of a "red-leg" like disease.~ It is best to keep the frogs in stationary water of a depth of 6 - 1 0 inches, and to change the water and clean the tanks twice weekly (after feeding). Amphibians are sensitive to both chlorine and chloramine in tap water. Although many communities use only chlorine in the water supply, many others have started to add both anhydrous ammonia and chlorine, which combine to form chloramine. Chloramine is very stable, slowly decomposing back to chlorine and ammonia. Neither reverse osmosis nor deionization removes all chloramine. The method of purification required depends on the local water supply treatment. If the water supply is treated only with chlorine, it can be removed by leaving the water in open containers exposed to the air for 48 hr. This can be accomplished more rapidly by
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bubbling air through the tanks with an air pump designed for an aquarium. The frogs can then be safely placed in the water. If the water supply is treated with chloramine, the above treatment will be ineffective owing to the stability of chloramine. The simplest method to remove chloramine is to use high purity carbon or Barnstead organic removal cartridge filters. Two activated charcoal filters should be used in series for safety, which will remove all detectable chloramine, chlorine, and ammonia. The water should be periodically tested at the outlet of each filter for chlorine and ammonia, and the filters replaced if either compound is present at detectable levels. This purification will result in water that can be used directly for the frogs without toxicity. An alternative method to remove chloramine is to use products which break it down to chlorine and ammonia. Many of these, such as Dechlor and Novaqua, are sold in pet stores for aquarium use. The free chlorine and ammonia must then be removed. The chlorine can be removed by allowing the water to sit exposed to the air for 48 hr, and the ammonia can be removed by filtration with zeolite. Alternatively, the ammonia can be converted to ammonium ions, which are not harmful to the frogs, by lowering the pH to 6.5.
Feeding Frogs only need to be fed about 2 times per week. Frogs bred in the wild will eat only meat, such as beef heart, liver, or similar food. A diet of meat exclusively is deficient in calcium and vitamin D, and so either the diet should be supplemented with trout chow or the meat should be soaked in a vitamin solution. A convenient meat diet is beef heart, which is easily available from most supermarket meat departments. Frogs bred in the laboratory will subsist quite well on a synthetic diet such as frog brittle, available from NASCO (Fort Atkinson, WI; Cat. No. SA5960MP for 5 pounds). In either case about 5 - 10 g of food per frog is dropped into the tank, and the frogs are allowed to eat for 4 - 6 h. At that point the tanks are cleaned to remove residual food and waste, and the frogs are placed in fresh water.
Diseases and Disorders Xenopus are quite hardy both in the wild and in the laboratory environment, with only a few percent mortality per year under normal laboratory conditions. The most notable disorder is red leg, in which underparts of the body and the inside of the mouth become red and swollen. This is due to an accumulation of blood. The disease can be treated by immersing the flogs in a 500 mg/ml solution of streptomycin and penicillin for a few days; otherwise, it is usually fatal. ~ Frogs which are kept in an environment with running water can suffer from a disease like red leg owing to hemolysis.
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Xenopus are very sensitive to antibiotics. Sulfonamides are lethal, and penicillin or streptomycin are harmful if injected. On the other hand, the frogs are protected by a natural peptide antimicrobial, magainin. 9 Magainin is a family of closely related peptides which inhibit the growth of numerous species of bacteria and fungi and cause lysis of protozoa by osmotic disruption. In the laboratory it functions to reduce the incidence of infection, particularly following surgery for oocyte removal. Preparation of Oocytes
Methods of Anesthesia The most commonly used anesthetic for Xenopus laevis has been MS222, the methane sulfonate salt of 3-aminobenzoic acid ethyl ester (also called tricaine, Sigma, St. Louis, MO; Cat. No. A-5040)J ° MS-222 is effective at a concentration as low as 0.15% and may be used safely to 0.35%. The compound dissolves easily in water and can be used multiple times by storing it at 4 °. The frog is placed in a small container containing 0.2% MS-222 for about 10-15 min, after which the extent of anesthesia should be tested. One test is to see if the frog can right itself after being placed upside down. The effectiveness of MS-222 appears to vary with different frogs, and sometimes it can require 30 min or more for adequate anesthesia. This can be overcome by using higher levels of MS-222, but concentrations greater than 0.35% can be debilitating to the frogs. The effect can also be enhanced by using the MS-222 at 4 °, but the anesthesia may wear off as the frog warms to room temperature. An alternative anesthetic that is effective on Xenopus is benzocaine (ethyl p-aminobenzoate, Sigma, Cat. No. E- 1501). u This compound is a reversible blocker of sodium channels and is used as a local anesthetic in mammals. It is as effective an anesthetic as MS-222 in frogs and other amphibians, and is more consistent in its effect. Benzocaine is effective on Xenopus at a concentration of 0.03%. Because it dissolves very slowly in water, a 100X stock (3%) should be made in 100% ethanol. The stock is then diluted 1 : 100 with water for immersion of the frogs, and the 1% residual ethanol causes no problems. Solutions of benzocaine are stable for at least 2 weeks at room temperature, and longer at 4 °. The frog is 9 M. Zasloff, Proc. Natl. Acad. Sci. U.S.A. 8,t, 5449 (1987). ~oV. Hamburger, "A Manual of Experimental Embryology." Univ. of Chicago Press, Chicago, Illinois, 1960. H R. B. Borgens, M. E. MeGinnis, J. W. Variable, Jr., and E. S. Miles, J. Exp. Zool. 231,249 (1984).
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anesthetized as with MS-222, which should take about 10-15 min and should be tested the same way. Following anesthesia and surgical removal of the ovaries, the frog should be allowed to recover in a small container of water. As frogs must breathe in air, it is possible to drown them by immersion in water that is too deep while the frog is still anesthetized. When the frog has completely recovered (30-60 min), it can be safely returned to the colony.
Removal of Oocytes When the frog is fully anesthetized, it should be placed on its back on a clean surface. A small incision about 1 cm long stretching diagonally from medial to lateral toward the head is made in the abdomen. Because the skin is extremely tough, it helps to use a sharp needle to pierce the skin, after which scissors can be used for the incision. Cut through both the skin and the underlying fascia, after which the ovaries should be visible. Remove as many oocytes as required by pulling out the lobes of the ovary with a pair of forceps, and using a pair of scissors to cut them. Place the oocytes in a 60-ram tissue culture dish containing calcium-free OR2 medium (see Appendix I). After a sufficient number of oocytes have been removed, replace the remaining ovary into the abdomen with the forceps and close the incision with one stitch using 5-0 silk. The entire operation should be done under clean conditions, but a sterile surgical procedure is not necessary because of the magainin antimicrobial peptides secreted from the skin. 9,12This allows a frog to be returned to the water immediately after surgery with a very low likelihood of infection developing. Healing generally occurs without gross inflammation or cellular reaction. It is possible to go back to the same frog and remove oocytes multiple times, but a deterioration in oocyte quality is sometimes seen after multiple surgeries. To keep track of individual frogs they can be marked by various means, including toe clipping, the tying of colored threads between the toes, or tattooing? 3 The first two methods generally work poorly because it is difficult to distinguish frogs in a large tank, and the threads can easily fall off. For tattooing, use 4 N HC1 to draw a number on the back of the frog while it is still anesthetized after removing the oocytes. The HC1 is left on for 1- 2 min, after which the frog is returned to water for recovery from anesthesia. An alternative is to use 0.5% amido black in 7% acetic acid. ~ 12 B. A. Berkowitz, C. L. Bevins, and M. A. Zasloff, Biochem. PharmacoL 39, 625 (1990). 13j. B. Gurdon, in "Methods in Developmental Biology" (F. H. Wilt and N. K. Wessells, eds.), p. 75. Thomas Y. Crowell, New York, 1967.
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While the tattooing method does theoretically provide an opportunity for infection, this is rarely seen. Frogs labeled by this method are easily distinguished in large tanks.
Removal of Follicle Cells Oocytes can be injected with follicle cells around them. This is technically more difficult, because the follicle cells are harder to pierce with the needle and it is time-consuming to separate out individual oocytes. However, there are some responses measured in oocytes that either depend on the presence of follicle cells or are actually occurring in the follicle cells, such as the opening of K ÷ channels in response to cyclic nucleotides. ~4 If these responses are not important to the study, then it is best to remove the follicle cells before injection. This can be done either manually or through the use of collagenase. Collagenase has the advantage of being able to defolliculate large numbers (thousands) of oocytes in a single step. This is essential for large-scale injections. However, overtreatment with collagenase significantly reduces oocyte viability. To avoid collagenase treatment, use a pair of forceps to pull apart the individual oocytes gently. These can then be manually defolliculated using forceps and scissors or with poly(L-lysine)-coated slides, as described by Miledi and W o o d w a r d . 14 Alternatively, the oocytes can be injected immediately with the follicle cells attached. If collagenase is to be used, the extent of treatment should be carefully monitored for removal of follicle cells. Use collagenase at a concentration of 0.5 units/ml, which generally corresponds to 2 mg/ml for most lots of collagenase. It is a good idea to test each lot of collagenase before largescale use, since different lots can vary in effectiveness and toxicity. For treatment, gently tease apart the oocytes while they are in calcium-free OR2 in a tissue culture dish. Use a pair of forceps, but be careful not to lyse too many oocytes. The goal is to separate the clumps of oocytes sufficiently to allow the collagenase to work. The oocytes are then incubated in a solution of collagenase in OR2 at 20 °. Incubating the oocytes in a test tube on a rotator allows better mixing than using a tissue culture dish. After about 90 min replace the solution with fresh collagenase, and remove the oocytes after another 60-90 min. Monitor the treatment carefully by removal of oocytes, and stop the treatment before all of the cells have been fully defolliculated to prevent toxicity. Collagenase is particularly damaging to oocytes if the incubation is carried out in the presence of calcium, which activates proteases. To prevent this wash the oocytes before collagenase treatment in calcium-free I,LR. Miledi and R. M. Woodward, J.
Physiol. (London) 416, 601 (1989).
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OR2, and then wash them extensively (4-6 times) after collagenase treatment in calcium-free OR2 before transferring them to a calcium-containing solution like ND96. Suitable sources of coUagenase are BoehringerMannheim (Indianapolis, IN; Cat. No. 1088 793) and Sigma (type IA, Cat. No. C-9891). Some researchers use only partial collagenase treatment (minutes to an hour) to maintain oocyte viability, after which the residual follicle cells are removed manually.
Selection of Oocytesfor Injection After collagenase treatment and OR2 washing, the oocytes should be transferred to incubation solution (see below). At this point it is a good idea to select the healthiest oocytes and transfer them to a fresh dish in incubation solution. Death and lysis of many oocytes are inevitable in the original dish owing to the large number of oocytes. This quickly results in a hypertonic solution, which is harmful to the otherwise healthy oocytes. After selection the oocytes should be maintained for a few hours before injection to allow any additional oocyte death to occur. There is no disadvantage to incubating the oocytes overnight before injection. Oocytes from Xenopus laevis can be classified into six stages of development on the basis of anatomy, with stage I being the earliest stage. 15 Stage I oocytes are about 50- 100/~m in diameter and appear transparent. Stage II oocytes are 300-450 #m and appear either translucent or white, depending on the extent of development. Stage III oocytes are 450-600 ~m and can be distinguished by the appearance of pigmentation uniformly throughout the surface. Stage IV oocytes are 600- 1000/tm, with differentiated hemispheres and a very dark brown animal hemisphere. Stage V oocytes are 1000- 1200/tm, with the hemispheres clearly delineated and a lightening in color of the animal hemisphere. Stage VI oocytes are the most mature, 1200-1300 gm, and can be distinguished by an unpigmented equatorial band between the two hemispheres. The choice of which stage oocytes to inject will depend on the purposes of the experiment. For most experiments stage V oocytes are optimal because they are large, can easily be injected with up to 100 nl of solution, and have significant translational capacity. However, the large size is reflected in a very large membrane capacitance (100-200 nF), so that voltage clamp recordings from these oocytes will demonstrate a long capacitive transient (1 - 2 msec). Stage II or III oocytes can also be used successfully for injection and expression, and the smaller size of these oocytes will result in a faster clamp settling time) 6 However, they can only be injected with 1~j. N. Dumont, J. Morphol. 136, 153 (1972). 16 D. S. Krafte and H. A. Lester, J. Neurosci. Methods 26, 211 (1989).
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about 20 nl of solution, and this must be performed using a more elaborate injection device such as those described below for nuclear injection. Injection of Oocytes
Preparation of RNA or DNA for Injection RNA or DNA for injection should be precipitated with ethanol at least once to remove excess salt and detergents like sodium dodecyl sulfate (SDS). These can have a markedly deleterious effect on the health of the oocytes. After precipitation the RNA should be resuspended at an appropriate concentration in either water or dilute Tris-HC1 (1 raM, pH 6.5). The low pH of RNA solutions in water can be slightly more toxic to the oocytes than a buffered solution. The appropriate concentration depends on the source of the RNA and varies from 10 mg/ml for total rat brain RNA to 10 gg/ml for in vitro transcribed RNA. At the very low concentrations it is advisable to use carrier RNA (such as yeast tRNA) to prevent losses from RNA sticking to the glass injection needles, but this is not essential.
Cytoplasmic Injection Cytoplasmic injections in oocytes can be performed with a very simple and inexpensive injection device? 7 This apparatus uses a Drummond 10-/tl microdispenser (oocyte injector, Drummond, Broomall, PA, Cat No. 3-00-510-X) attached to a three-dimensional coarse micromanipulator such as a Brinkmann (Westbury, NY) MM-33. The attachment requires a custom-made ring that mounts onto the micromanipulator, allowing the microdispenser to be held in place by a set screw. The oocytes are placed in ND96 in a 35-mm tissue culture dish under a stereomieroscope. To keep the oocytes from moving, polypropylene mesh (Sepetra/Mesh PP, Fisher, Cat. No. 08-670-185) can be glued to the bottom of the dish. Injection needles are made by using a pipette puller to draw out the glass bores that are normally used with the Drummond microdispenser. The standard 4 inch bores work quite well with a vertical puller, but ifa horizontal puller is used it requires 8 inch bores available from Drummond to make two injection needles. After pulling, the needles are broken offat a tip diameter of 2 0 - 40 am, as measured with a reticle under a dissecting microscope. To attach the needles to the dispenser, add about 5 m m of fight mineral oil into the needle near the large opening, then insert the bore onto the dispenser plunger all the way. The plunger will force the oil toward the tip t7 R. Contreras, H. Cheroutre, and W. Fiers, Anal Biochem. 113, 185 (1981).
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of the needle, which should result in a complete seal of oil between the metal plunger and the injection needle tip. Before drawing the RNA into the needle the RNA solution should be centrifuged in a microcentrifuge tube for about 60 sec to pellet insoluble debris. This reduces the likelihood of needle clogging. About 2#1 of RNA solution is then placed on a tissue culture dish, and the solution is drawn into the Drummond microdispenser. It is advisable to watch the RNA being drawn into the needle through the microscope to be sure that the needle has not clogged. Once the needle is filled with RNA it can be positioned over each oocyte and gently lowered until it pierces the oocyte. Up to 100 nl can then be injected into each oocyte by turning the knob on the microdispenser. By this method it is possible to inject 20 oocytes with one sample in a few minutes. Although the same needle can then be used for additional RNA samples, using a new needle for each sample prevents cross-contamination and makes needle clogging less likely. An alternative injection device has recently been introduced by Drummond (Nanoject, Cat. No. 3-00-203-X). This consists of a motor-driven dispenser similar to the 10-/d microdispenser and includes controls for continuously drawing up solution, continuously forcing out solution, and a momentary switch for injecting 47 nl with each push of a button. This has the advantage that injections can be performed without ever looking away from the oocytes. The disadvantages are that the unit is more expensive and can malfunction more easily. An injector with adjustable volume is also available (Cat. No. 3-00-203-XV).
Nuclear Injection Nuclear injections require a more sophisticated injection apparatus, as the maximum volumes that can be injected are about 10-20 hi. The Picopump from World Precision Instruments (Sarasota, FL) and the Picospritzer from General Valve (Fairfield, NJ) use air pressure for injection, and the Nanopump from World Precision Instruments uses a metal plunger for injection. The air pressure devices can be used for smaller volumes, but they must be calibrated for each needle. The Nanopump is slower and requires a tight seal of oil between the plunger and RNA. In either case the micromanipulator is the same, only the injection volume is controlled by either the injection pressure or the time of injection. All these instruments can, of course, be used for cytoplasmic injection also. For nuclear injection it is necessary to align the nuclei on the top surface of the oocytes so that the needle can be inserted into the appropriate location. This can be accomplished by centrifuging the oocytes, since the nucleus is less dense than the cytoplasm and rises to the top surface. Microwell dishes from Irvine Scientific (Cat. No. 1-63118) work well for
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this purpose. Single oocytes are placed in each well in ND96 with the pigmented side facing up. The dish can be placed on a test tube holder in a tabletop centrifuge, such as a Beckman GPR (Fullerton, CA), for centrifugation. It is a good idea to perform a test spin with about 10 oocytes to determine the centrifugation conditions. Conditions which are a reasonable starting point are 1000 g for 13 rain. Examine each oocyte for the presence of the nucleus at the top surface, which will appear as a lighter color in the pigmentation. Optimal conditions result in a clear area of decreased pigmentation in all of the oocytes without a significant number of dead ooctyes. Both the time and centrifugal force can be varied to improve oocyte viability. The oocytes should be injected within 30-60 rain after centrifugation (if they sit for too long in the microwell dish a significant number will probably die). The injection needles must be smaller in diameter than those used on the Drummond injectors for cytoplasmic injection. Suitable glass is R6, 0.63 m m OD by 0.20 m m ID by 10 cm long, also available from Drummond. The needles are pulled as usual and broken off at about 30/~m outer diameter. If a pressure injection sytem is used, the needles must then be calibrated. This is easily accomplished by injecting an aqueous dye solution (Injection Dye, see Appendix I) into glycerol, which results in a sphere of fluid. The diameter of the sphere is measured with the microscope reticle, and the volume can be directly determined using a standard graph of volume versus sphere diameter ( V = ~tda/6; 10 nl is approximately equivalent to a diameter of 270 am). The injection dye is also useful to include with the DNA solution, as this will serve as an indicator of successful nuclear injection. The DNA or RNA solution should be drawn into the injection needle with sufficient vaccum to result in about 1 fll drawn up in 60 sec. The ejection pressure or time should then be varied to achieve an appropriate injection volume. Using a Picopump, approximate conditions for injecting 10-nl volumes are a vacuum of 1015 inches mercury and an ejection pressure of 10-14 psi, with a 1-sec injection time. It is probably best to adjust the ejection pressure and keep the time constant, as oocytes do not tolerate extremely rapid injection. Incubation of Oocytes After the oocytes have been defolliculated or injected they should be maintained in a relatively isoosmotic solution containing calcium. A wide variety of solutions have been used for this purpose, including Barth's, 4 OR2 with calcium,~S normal frog Ringer's solution,19 L-15 medium, 2° and ts R. A. Wallace, D. W. Jared, J. N. Dumont, and M. W. Sega, J. Exp. Zool. 184, 321 (1973).
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ND962~ (recipes are given in Appendix I). ND96 is essentially OR2 which is slightly hypertonic (sodium concentration 96 compared to 82.5 mM), which may be beneficial when injecting the oocytes. In addition, the incubation medium should be supplemented with sodium pyruvate (550 mg/liter) as a carbon source22 and gentamicin (100 #g/ml) to prevent bacterial contamination.23 An alternative antibiotic mix that is effective is a combination of 100 U/ml penicillin and 100 #g/ml streptomycin.24 Theophylline can be added to a concentration of 0.5 m M to inhibit phosphodiesterase and keep cAMP level high, which will prevent maturation. This is generally not necessary, however, because cAMP levels are normally high in oocytes.25 The oocytes should be incubated in 35-mm diameter tissue culture dishes (60-ram diameter dishes for large numbers) on a rotator at slow speed in a 20 ° controlled temperature incubator. Temperature fluctuations, particularly heat, can both be damaging to oocyte viability and result in erratic time courses of expression. The healthy ooctyes should be transferred to fresh dishes with new ND96 each day, and more often if there is any oocyte death. This is particularly important shortly after injection, as dying oocytes release salts which make the solution hypertonic and toxic to the remainder. The time course of expression varies with the type of RNA injected. Detectable expression of sodium channels can be observed 24 hr after injection of RNA made in vitro from a clone when the oocytes are incubated at 20 ° (lower temperatures result in a longer delay). Expression of this channel reaches maximal levels by about 2 days and does not noticeably decrease at least until 6 days postinjection. Appendix I: Recipes OR2--calcium-free 82.5 mM NaC1 2mM KCI 1 mM MgC12 5 mM HEPES,pH 7.5 with NaOH
19C. Methfessel,V. Witzemann,T. Takahashi, M. Mishina, S. Numa, and B. Sakmann, Pfluegers Arch. 407, 577 (1986). 2oR. A. Wallace,Z. Misulovin,D. W. Jared, and H. S. Wiley,GameteRes. 1, 269 (1978). 21j. p. Leonard,J. Nargeot,T. P. Snutch,N. Davidson,and H. A. Lester,J. Neurosci, 7, 875 (1987). 22j. j. Eppigand J. N. Dumont,In Vitro 12, 418 (1976). 23R. A. Wallaceand Z. Misulovin,Proc. Natl. Acad. Sci. U.S.A. 75, 5534 (1978). 24R. A. Laskey,J. CellSci. 7, 653 (1970). 25M. F. Cicirelliand L. D. Smith,Dev. Biol. 108, 254 (1985).
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ND96 96 mM NaCI 2 mM KC1 1.8 mM CaCl2 1 mM MgC12 5 mM HEPES, pH 7.5 with NaOH Modified Barth's solution 88 mM NaCl 1 mM KC1 2.4 mM NaHCO3 20 mM HEPES, pH 7.5 0.82 mM MgSO4 0.33 mM Ca(NO3)2 0.41 mM CaCI2 Normal frog Ringer's solution 115 mM NaC1 2.5 mMKC1 1.8 mM CaC12 10 mM HEPES, pH 7.2 L-15 70% Leibovitz's L-15 medium (available from GIBCO, Grand Island, NY) 10 mMHEPES, pH 7.5 100X Injection Dye 0.2 M Tris-HCl, pH 7.5 1 M NaC1 4% Trypan blue (Use 10× for needle calibration and IX for DNA samples)
Appendix II: Suppliers ofXenopus laevis United States Suppliers NASCO, 901 Janesville Ave., Fort Atkinson, WI 53538, (414) 563-2446. Ordering Information: LM535MP, adult females, 9-10.5 cm @ $20.60; LM531P, guaranteed ooeyte-positive females; LM535WC, adult females, 9-10.5 cm @ $17.20 (wild-caught); LM531WC, guaranteed oocyte-positivefemales (wild-caught). Notes: The first two catalog numbers are for laboratory-reared frogs. The last two are for wild-caught, laboratory-conditioned frogs (a recent addition to the catalog). The 9-10.5 cm adult females are generally the best source of mature oocytes, as these frogs are guaranteed to be large and have always been ooeyte positive. Xenopus I, 716 Northside, Ann Arbor, MI 48105, (313) 426-2083. Ordering Information: 4216, adult females, ooeyte positive, @ $13.90. Notes: Frogs captured in the wild. Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215, (800) 334-5551, (919) 584-0381. Ordering Information: L1570, adult females @ $18.79. Notes: Laboratory-reared frogs.
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European Suppliers Xenopus Ltd., Holmesdaie Nursery, Mid Street, South Nutfield, RedhiU, Surrey RH1 4JY, England, 44-73-782-2687. Ordering Information: Very large, mature females. Notes: Either laboratory-reared or wild frogs. Dipl. Biol.-Dipl. Ing. Horst KAMer Institut f'tir Entwicklungsbiologle Kollaustrasse 113b D-2000 Hamburg 61 Germany 40-587675 Ordering Information: X. laevisor X. laevisalbinos Notes: Either laboratory-reared or wild frogs.
Japanese Suppliers Nippon Life Science, ordering address: 1-1-32 Shiba-Daimon, Minatoku, Tokyo 105; company address: Denmacho 19, Hamamatsu City, Shizuoka, Japan. Seibu Department Stores Tokyo, Japan Notes: Xenopus can be purchased in the departmem stores in Japan.
South African Supplier South African Snake Farm, P.O. Box 6, Fish Hock, Cape Province, Republic of South Africa. Notes: Frogs can be ordered directly from South Africa for air shipment anywhere in the world.
Acknowledgments The author is a LuciUe P. Markey Scholar, and work in his laboratory is supported by grants from the U.S. National Institutes of Health (NS-26729), the Lucille P. Markey Charitable Trust, and the March of Dimes Basil O'Connor Starter Scholar Program.
[ 16] Preparation of RNA for Injection into Xenopus Oocytes By ALAN L. GOLDIN a n d KATUMI SUMIKAWA Introduction
Xenopus oocytes are n o w a p o p u l a r system for the expression a n d characterization o f functional n e u r o t r a n s m i t t e r receptors a n d ion channels. O o c y t e s are injected with m R N A s extracted f r o m tissues or syntheMETHODS IN ENZYMOLOGY, VOL. 207
Copyright© 1992by AcademicPress,Inc. All fightsof reproductionin any formr--~erved.
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[15] Maintenance of Xenopus laevis and Oocyte Injection By ALANL. GOLDIN Introduction
Xenopus laevis, the South African dawed frog, is a member of the anuran family Pipidae. There are six species of Xenopus, all indigenous to Africa, but Xenopus laevis is the only one commonly used in laboratory research.~ Xenopus laevis frogs are distinguished by the presence of three toes with claws on each hind foot of the animal, hence the name, which means "clawed frog." There are four subspecies ofXenopus laevis, termed X. laevis laevis, X. laevis petersi, X. laevis victorianus, and X. laevis borealis. 1 All Xenopus are aerobic but entirely aquatic, so there is an absolute requirement for breathing air but no necessity for any land-based existence. Xenopus are very susceptible to desiccation, and they can die from being out of the water for a few hours. Xenopus frogs are normally darkly pigmented on the dorsal side, but their skin contains chromatophores which rapidly change color according to the surrounding environment. The frog secretes both a mucouslike substance and a toxic substance with properties like epinephrine. These are most likely defense mechanisms for the frog, but they are not harmful to humans when handling the frogs. Xenopus laevis was first used experimentally in a clinical setting as a pregnancy test. Female frogs were injected with urine from a possibly pregnant woman, which resulted in egg laying only if the urine contained human chorionic gonadotropin (hCG), an indication of pregnancy. Although the frogs are no longer used for this purpose, they are still widely used as an experimental system in developmental biology.2 However, the most common use today of Xenopus laevis has been for their oocytes. Gurdon et aL originally demonstrated that Xenopus oocytes can be used to express exogenous mRNA species when microinjected into the cytoplasm) Many investigators have sinced used this property to study the expression and translation of a large number of molecules (for reviews, see Rcfs. 4-8). J. B. Gurdon and H. R. Woodland, in "Handbookof Genetics"(R. C. King, ed.), p. 35. Plenum, New York, 1975. 2I. B. Dawidand T. D. Sargent,Science240, 1443(1988). 3j. B. Gurdon, C. D. Lane, H. R. Woodlandand G. Marbaix, Nature (London) 233, 177 (1971). 4j. B. Gurdonand M. P. Wickens,this series, Vol. 101,p. 370. s A. Colman,in "Transcriptionand Translation--A PracticalApproach"(B. D. Hamesand S. J. Higgins,(Ms.),p. 271. IRL Press, Oxford, 1984. METHODS IN ENZYMOL(X}Y, VOL. 207
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reservod.
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Sources of Xenopus Frogs Although Xenopus laevis is indigenous only to South Africa, the frogs are now available from a number of different suppliers throughout the world (see Appendix II for a listing). There are two major types of frog suppliers, those that capture and sell frogs reared in the wild and those that sell frogs bred in their own facilities. Frogs bred in the laboratory are generally more consistent and can be fed a synthetic diet available from various suppliers (see below). On the other hand, frogs captured in the wild may be less susceptible to disease than inbred frogs .1 They will not eat a synthetic diet such as frog brittle, however, and in fact may die of starvation if kept on such a diet. They must be fed a natural diet consisting of liver, heart, or similar material (see below). Both laboratoryreared and wild-reared frogs can be used successfully for the production of oocytes. The specifics of ordering frogs for oocyte use vary with each supplier, but it is generally a good idea to request the largest and most mature females for the purpose of obtaining stage V oocytes. These large frogs also contain a significant number of less mature oocytes, so that it is possible to obtain oocytes of all stages from them. In addition, it is advisable to request shipping with cold packs during the summer months to prevent extremes of temperature variation. To avoid this problem it may be helpful to have sufficient frogs shipped prior to the hot months of summer. Once the frogs have arrived it is a good idea to let them acclimatize for about 2 weeks before use. In addition, because of the widespread use of Xenopus frogs for oocyte injection experiments, there have been recent shortages, particularly from the suppliers that sell laboratory-bred frogs. To synchronize oocyte development, the frogs can be injected with hCG, which induces ovulation and egg laying, after which oocyte development starts over approximately synchronously. An appropriate dose is 400-500 IU, which should induce egg-laying in about 12 hr? Many suppliers offer flogs which have been injected with hCG. If hCG-injected frogs are to be ordered, however, be sure to request that the frogs be kept for 2 - 3 months after injection to allow sufficient time for mature oocytes to develop. Uninjected frogs generally are just as good a source of mature oocytes as injected ones, so that hCG injection is not usually necessary.
6 H. Soreq, Crit. Rev. Biochem. 18, 199 (1985). 7 T. P. Snutch, Trends Neurosci. 11, 250 (1988). 8 N. Dascal, Crit. Rev. Biochem. 22, 317 (1987).
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M a i n t e n a n c e of Frogs
Environmentfor Frogs Xenopus can tolerate a wide range of temperature fluctuations, but the quality of oocytes diminishes markedly following temperature shifts. The frogs should therefore be housed in a temperature-controlled environment at approximately 20 °. Sufficient air conditioning is especially important, as heat seems to be more detrimental than cold. In addition, the frogs should be maintained in a constant light-dark cycle of 12 hr each. This helps to prevent the seasonal variability found with Xenopus in the wild, although oocyte quality generally does diminish during the summer months. A stable environment will help to ensure that the oocytes are still usable, however. The containers for the frogs need not be particularly sophisticated. Large polypropylene tanks or aquarium tanks both work well. The advantage of polypropylene is that it keeps the frogs more isolated from the external environment, but aquarium glass can be painted black to achieve the same effect. This is beneficial since the frogs are easily startled by movement or light. It is important to have a tight-fitting lid with holes for air, since the frogs can easily lift a lid and escape. Frogs are obligate air breathers, and must be able to reach the surface, so the level of water should not be greater than about 6 - 1 0 inches. Suitable containers are Nalgene tanks 18 by 12 by 12 inches high (Fisher, Pittsburgh, PA, Cat. No. 14-831-330), with holes drilled in the lids. Each of these containers can hold 6 - 8 frogs, with 2 - 3 liters of water per frog. Treatment of Water Xenopus are freshwater frogs that prefer stationary water. If running water is used to keep the water clean, it should be provided at a slow rate to prevent development of a "red-leg" like disease.~ It is best to keep the frogs in stationary water of a depth of 6 - 1 0 inches, and to change the water and clean the tanks twice weekly (after feeding). Amphibians are sensitive to both chlorine and chloramine in tap water. Although many communities use only chlorine in the water supply, many others have started to add both anhydrous ammonia and chlorine, which combine to form chloramine. Chloramine is very stable, slowly decomposing back to chlorine and ammonia. Neither reverse osmosis nor deionization removes all chloramine. The method of purification required depends on the local water supply treatment. If the water supply is treated only with chlorine, it can be removed by leaving the water in open containers exposed to the air for 48 hr. This can be accomplished more rapidly by
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bubbling air through the tanks with an air pump designed for an aquarium. The frogs can then be safely placed in the water. If the water supply is treated with chloramine, the above treatment will be ineffective owing to the stability of chloramine. The simplest method to remove chloramine is to use high purity carbon or Barnstead organic removal cartridge filters. Two activated charcoal filters should be used in series for safety, which will remove all detectable chloramine, chlorine, and ammonia. The water should be periodically tested at the outlet of each filter for chlorine and ammonia, and the filters replaced if either compound is present at detectable levels. This purification will result in water that can be used directly for the frogs without toxicity. An alternative method to remove chloramine is to use products which break it down to chlorine and ammonia. Many of these, such as Dechlor and Novaqua, are sold in pet stores for aquarium use. The free chlorine and ammonia must then be removed. The chlorine can be removed by allowing the water to sit exposed to the air for 48 hr, and the ammonia can be removed by filtration with zeolite. Alternatively, the ammonia can be converted to ammonium ions, which are not harmful to the frogs, by lowering the pH to 6.5.
Feeding Frogs only need to be fed about 2 times per week. Frogs bred in the wild will eat only meat, such as beef heart, liver, or similar food. A diet of meat exclusively is deficient in calcium and vitamin D, and so either the diet should be supplemented with trout chow or the meat should be soaked in a vitamin solution. A convenient meat diet is beef heart, which is easily available from most supermarket meat departments. Frogs bred in the laboratory will subsist quite well on a synthetic diet such as frog brittle, available from NASCO (Fort Atkinson, WI; Cat. No. SA5960MP for 5 pounds). In either case about 5 - 10 g of food per frog is dropped into the tank, and the frogs are allowed to eat for 4 - 6 h. At that point the tanks are cleaned to remove residual food and waste, and the frogs are placed in fresh water.
Diseases and Disorders Xenopus are quite hardy both in the wild and in the laboratory environment, with only a few percent mortality per year under normal laboratory conditions. The most notable disorder is red leg, in which underparts of the body and the inside of the mouth become red and swollen. This is due to an accumulation of blood. The disease can be treated by immersing the flogs in a 500 mg/ml solution of streptomycin and penicillin for a few days; otherwise, it is usually fatal. ~ Frogs which are kept in an environment with running water can suffer from a disease like red leg owing to hemolysis.
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Xenopus are very sensitive to antibiotics. Sulfonamides are lethal, and penicillin or streptomycin are harmful if injected. On the other hand, the frogs are protected by a natural peptide antimicrobial, magainin. 9 Magainin is a family of closely related peptides which inhibit the growth of numerous species of bacteria and fungi and cause lysis of protozoa by osmotic disruption. In the laboratory it functions to reduce the incidence of infection, particularly following surgery for oocyte removal. Preparation of Oocytes
Methods of Anesthesia The most commonly used anesthetic for Xenopus laevis has been MS222, the methane sulfonate salt of 3-aminobenzoic acid ethyl ester (also called tricaine, Sigma, St. Louis, MO; Cat. No. A-5040)J ° MS-222 is effective at a concentration as low as 0.15% and may be used safely to 0.35%. The compound dissolves easily in water and can be used multiple times by storing it at 4 °. The frog is placed in a small container containing 0.2% MS-222 for about 10-15 min, after which the extent of anesthesia should be tested. One test is to see if the frog can right itself after being placed upside down. The effectiveness of MS-222 appears to vary with different frogs, and sometimes it can require 30 min or more for adequate anesthesia. This can be overcome by using higher levels of MS-222, but concentrations greater than 0.35% can be debilitating to the frogs. The effect can also be enhanced by using the MS-222 at 4 °, but the anesthesia may wear off as the frog warms to room temperature. An alternative anesthetic that is effective on Xenopus is benzocaine (ethyl p-aminobenzoate, Sigma, Cat. No. E- 1501). u This compound is a reversible blocker of sodium channels and is used as a local anesthetic in mammals. It is as effective an anesthetic as MS-222 in frogs and other amphibians, and is more consistent in its effect. Benzocaine is effective on Xenopus at a concentration of 0.03%. Because it dissolves very slowly in water, a 100X stock (3%) should be made in 100% ethanol. The stock is then diluted 1 : 100 with water for immersion of the frogs, and the 1% residual ethanol causes no problems. Solutions of benzocaine are stable for at least 2 weeks at room temperature, and longer at 4 °. The frog is 9 M. Zasloff, Proc. Natl. Acad. Sci. U.S.A. 8,t, 5449 (1987). ~oV. Hamburger, "A Manual of Experimental Embryology." Univ. of Chicago Press, Chicago, Illinois, 1960. H R. B. Borgens, M. E. MeGinnis, J. W. Variable, Jr., and E. S. Miles, J. Exp. Zool. 231,249 (1984).
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anesthetized as with MS-222, which should take about 10-15 min and should be tested the same way. Following anesthesia and surgical removal of the ovaries, the frog should be allowed to recover in a small container of water. As frogs must breathe in air, it is possible to drown them by immersion in water that is too deep while the frog is still anesthetized. When the frog has completely recovered (30-60 min), it can be safely returned to the colony.
Removal of Oocytes When the frog is fully anesthetized, it should be placed on its back on a clean surface. A small incision about 1 cm long stretching diagonally from medial to lateral toward the head is made in the abdomen. Because the skin is extremely tough, it helps to use a sharp needle to pierce the skin, after which scissors can be used for the incision. Cut through both the skin and the underlying fascia, after which the ovaries should be visible. Remove as many oocytes as required by pulling out the lobes of the ovary with a pair of forceps, and using a pair of scissors to cut them. Place the oocytes in a 60-ram tissue culture dish containing calcium-free OR2 medium (see Appendix I). After a sufficient number of oocytes have been removed, replace the remaining ovary into the abdomen with the forceps and close the incision with one stitch using 5-0 silk. The entire operation should be done under clean conditions, but a sterile surgical procedure is not necessary because of the magainin antimicrobial peptides secreted from the skin. 9,12This allows a frog to be returned to the water immediately after surgery with a very low likelihood of infection developing. Healing generally occurs without gross inflammation or cellular reaction. It is possible to go back to the same frog and remove oocytes multiple times, but a deterioration in oocyte quality is sometimes seen after multiple surgeries. To keep track of individual frogs they can be marked by various means, including toe clipping, the tying of colored threads between the toes, or tattooing? 3 The first two methods generally work poorly because it is difficult to distinguish frogs in a large tank, and the threads can easily fall off. For tattooing, use 4 N HC1 to draw a number on the back of the frog while it is still anesthetized after removing the oocytes. The HC1 is left on for 1- 2 min, after which the frog is returned to water for recovery from anesthesia. An alternative is to use 0.5% amido black in 7% acetic acid. ~ 12 B. A. Berkowitz, C. L. Bevins, and M. A. Zasloff, Biochem. PharmacoL 39, 625 (1990). 13j. B. Gurdon, in "Methods in Developmental Biology" (F. H. Wilt and N. K. Wessells, eds.), p. 75. Thomas Y. Crowell, New York, 1967.
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While the tattooing method does theoretically provide an opportunity for infection, this is rarely seen. Frogs labeled by this method are easily distinguished in large tanks.
Removal of Follicle Cells Oocytes can be injected with follicle cells around them. This is technically more difficult, because the follicle cells are harder to pierce with the needle and it is time-consuming to separate out individual oocytes. However, there are some responses measured in oocytes that either depend on the presence of follicle cells or are actually occurring in the follicle cells, such as the opening of K ÷ channels in response to cyclic nucleotides. ~4 If these responses are not important to the study, then it is best to remove the follicle cells before injection. This can be done either manually or through the use of collagenase. Collagenase has the advantage of being able to defolliculate large numbers (thousands) of oocytes in a single step. This is essential for large-scale injections. However, overtreatment with collagenase significantly reduces oocyte viability. To avoid collagenase treatment, use a pair of forceps to pull apart the individual oocytes gently. These can then be manually defolliculated using forceps and scissors or with poly(L-lysine)-coated slides, as described by Miledi and W o o d w a r d . 14 Alternatively, the oocytes can be injected immediately with the follicle cells attached. If collagenase is to be used, the extent of treatment should be carefully monitored for removal of follicle cells. Use collagenase at a concentration of 0.5 units/ml, which generally corresponds to 2 mg/ml for most lots of collagenase. It is a good idea to test each lot of collagenase before largescale use, since different lots can vary in effectiveness and toxicity. For treatment, gently tease apart the oocytes while they are in calcium-free OR2 in a tissue culture dish. Use a pair of forceps, but be careful not to lyse too many oocytes. The goal is to separate the clumps of oocytes sufficiently to allow the collagenase to work. The oocytes are then incubated in a solution of collagenase in OR2 at 20 °. Incubating the oocytes in a test tube on a rotator allows better mixing than using a tissue culture dish. After about 90 min replace the solution with fresh collagenase, and remove the oocytes after another 60-90 min. Monitor the treatment carefully by removal of oocytes, and stop the treatment before all of the cells have been fully defolliculated to prevent toxicity. Collagenase is particularly damaging to oocytes if the incubation is carried out in the presence of calcium, which activates proteases. To prevent this wash the oocytes before collagenase treatment in calcium-free I,LR. Miledi and R. M. Woodward, J.
Physiol. (London) 416, 601 (1989).
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OR2, and then wash them extensively (4-6 times) after collagenase treatment in calcium-free OR2 before transferring them to a calcium-containing solution like ND96. Suitable sources of coUagenase are BoehringerMannheim (Indianapolis, IN; Cat. No. 1088 793) and Sigma (type IA, Cat. No. C-9891). Some researchers use only partial collagenase treatment (minutes to an hour) to maintain oocyte viability, after which the residual follicle cells are removed manually.
Selection of Oocytesfor Injection After collagenase treatment and OR2 washing, the oocytes should be transferred to incubation solution (see below). At this point it is a good idea to select the healthiest oocytes and transfer them to a fresh dish in incubation solution. Death and lysis of many oocytes are inevitable in the original dish owing to the large number of oocytes. This quickly results in a hypertonic solution, which is harmful to the otherwise healthy oocytes. After selection the oocytes should be maintained for a few hours before injection to allow any additional oocyte death to occur. There is no disadvantage to incubating the oocytes overnight before injection. Oocytes from Xenopus laevis can be classified into six stages of development on the basis of anatomy, with stage I being the earliest stage. 15 Stage I oocytes are about 50- 100/~m in diameter and appear transparent. Stage II oocytes are 300-450 #m and appear either translucent or white, depending on the extent of development. Stage III oocytes are 450-600 ~m and can be distinguished by the appearance of pigmentation uniformly throughout the surface. Stage IV oocytes are 600- 1000/tm, with differentiated hemispheres and a very dark brown animal hemisphere. Stage V oocytes are 1000- 1200/tm, with the hemispheres clearly delineated and a lightening in color of the animal hemisphere. Stage VI oocytes are the most mature, 1200-1300 gm, and can be distinguished by an unpigmented equatorial band between the two hemispheres. The choice of which stage oocytes to inject will depend on the purposes of the experiment. For most experiments stage V oocytes are optimal because they are large, can easily be injected with up to 100 nl of solution, and have significant translational capacity. However, the large size is reflected in a very large membrane capacitance (100-200 nF), so that voltage clamp recordings from these oocytes will demonstrate a long capacitive transient (1 - 2 msec). Stage II or III oocytes can also be used successfully for injection and expression, and the smaller size of these oocytes will result in a faster clamp settling time) 6 However, they can only be injected with 1~j. N. Dumont, J. Morphol. 136, 153 (1972). 16 D. S. Krafte and H. A. Lester, J. Neurosci. Methods 26, 211 (1989).
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about 20 nl of solution, and this must be performed using a more elaborate injection device such as those described below for nuclear injection. Injection of Oocytes
Preparation of RNA or DNA for Injection RNA or DNA for injection should be precipitated with ethanol at least once to remove excess salt and detergents like sodium dodecyl sulfate (SDS). These can have a markedly deleterious effect on the health of the oocytes. After precipitation the RNA should be resuspended at an appropriate concentration in either water or dilute Tris-HC1 (1 raM, pH 6.5). The low pH of RNA solutions in water can be slightly more toxic to the oocytes than a buffered solution. The appropriate concentration depends on the source of the RNA and varies from 10 mg/ml for total rat brain RNA to 10 gg/ml for in vitro transcribed RNA. At the very low concentrations it is advisable to use carrier RNA (such as yeast tRNA) to prevent losses from RNA sticking to the glass injection needles, but this is not essential.
Cytoplasmic Injection Cytoplasmic injections in oocytes can be performed with a very simple and inexpensive injection device? 7 This apparatus uses a Drummond 10-/tl microdispenser (oocyte injector, Drummond, Broomall, PA, Cat No. 3-00-510-X) attached to a three-dimensional coarse micromanipulator such as a Brinkmann (Westbury, NY) MM-33. The attachment requires a custom-made ring that mounts onto the micromanipulator, allowing the microdispenser to be held in place by a set screw. The oocytes are placed in ND96 in a 35-mm tissue culture dish under a stereomieroscope. To keep the oocytes from moving, polypropylene mesh (Sepetra/Mesh PP, Fisher, Cat. No. 08-670-185) can be glued to the bottom of the dish. Injection needles are made by using a pipette puller to draw out the glass bores that are normally used with the Drummond microdispenser. The standard 4 inch bores work quite well with a vertical puller, but ifa horizontal puller is used it requires 8 inch bores available from Drummond to make two injection needles. After pulling, the needles are broken offat a tip diameter of 2 0 - 40 am, as measured with a reticle under a dissecting microscope. To attach the needles to the dispenser, add about 5 m m of fight mineral oil into the needle near the large opening, then insert the bore onto the dispenser plunger all the way. The plunger will force the oil toward the tip t7 R. Contreras, H. Cheroutre, and W. Fiers, Anal Biochem. 113, 185 (1981).
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of the needle, which should result in a complete seal of oil between the metal plunger and the injection needle tip. Before drawing the RNA into the needle the RNA solution should be centrifuged in a microcentrifuge tube for about 60 sec to pellet insoluble debris. This reduces the likelihood of needle clogging. About 2#1 of RNA solution is then placed on a tissue culture dish, and the solution is drawn into the Drummond microdispenser. It is advisable to watch the RNA being drawn into the needle through the microscope to be sure that the needle has not clogged. Once the needle is filled with RNA it can be positioned over each oocyte and gently lowered until it pierces the oocyte. Up to 100 nl can then be injected into each oocyte by turning the knob on the microdispenser. By this method it is possible to inject 20 oocytes with one sample in a few minutes. Although the same needle can then be used for additional RNA samples, using a new needle for each sample prevents cross-contamination and makes needle clogging less likely. An alternative injection device has recently been introduced by Drummond (Nanoject, Cat. No. 3-00-203-X). This consists of a motor-driven dispenser similar to the 10-/d microdispenser and includes controls for continuously drawing up solution, continuously forcing out solution, and a momentary switch for injecting 47 nl with each push of a button. This has the advantage that injections can be performed without ever looking away from the oocytes. The disadvantages are that the unit is more expensive and can malfunction more easily. An injector with adjustable volume is also available (Cat. No. 3-00-203-XV).
Nuclear Injection Nuclear injections require a more sophisticated injection apparatus, as the maximum volumes that can be injected are about 10-20 hi. The Picopump from World Precision Instruments (Sarasota, FL) and the Picospritzer from General Valve (Fairfield, NJ) use air pressure for injection, and the Nanopump from World Precision Instruments uses a metal plunger for injection. The air pressure devices can be used for smaller volumes, but they must be calibrated for each needle. The Nanopump is slower and requires a tight seal of oil between the plunger and RNA. In either case the micromanipulator is the same, only the injection volume is controlled by either the injection pressure or the time of injection. All these instruments can, of course, be used for cytoplasmic injection also. For nuclear injection it is necessary to align the nuclei on the top surface of the oocytes so that the needle can be inserted into the appropriate location. This can be accomplished by centrifuging the oocytes, since the nucleus is less dense than the cytoplasm and rises to the top surface. Microwell dishes from Irvine Scientific (Cat. No. 1-63118) work well for
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this purpose. Single oocytes are placed in each well in ND96 with the pigmented side facing up. The dish can be placed on a test tube holder in a tabletop centrifuge, such as a Beckman GPR (Fullerton, CA), for centrifugation. It is a good idea to perform a test spin with about 10 oocytes to determine the centrifugation conditions. Conditions which are a reasonable starting point are 1000 g for 13 rain. Examine each oocyte for the presence of the nucleus at the top surface, which will appear as a lighter color in the pigmentation. Optimal conditions result in a clear area of decreased pigmentation in all of the oocytes without a significant number of dead ooctyes. Both the time and centrifugal force can be varied to improve oocyte viability. The oocytes should be injected within 30-60 rain after centrifugation (if they sit for too long in the microwell dish a significant number will probably die). The injection needles must be smaller in diameter than those used on the Drummond injectors for cytoplasmic injection. Suitable glass is R6, 0.63 m m OD by 0.20 m m ID by 10 cm long, also available from Drummond. The needles are pulled as usual and broken off at about 30/~m outer diameter. If a pressure injection sytem is used, the needles must then be calibrated. This is easily accomplished by injecting an aqueous dye solution (Injection Dye, see Appendix I) into glycerol, which results in a sphere of fluid. The diameter of the sphere is measured with the microscope reticle, and the volume can be directly determined using a standard graph of volume versus sphere diameter ( V = ~tda/6; 10 nl is approximately equivalent to a diameter of 270 am). The injection dye is also useful to include with the DNA solution, as this will serve as an indicator of successful nuclear injection. The DNA or RNA solution should be drawn into the injection needle with sufficient vaccum to result in about 1 fll drawn up in 60 sec. The ejection pressure or time should then be varied to achieve an appropriate injection volume. Using a Picopump, approximate conditions for injecting 10-nl volumes are a vacuum of 1015 inches mercury and an ejection pressure of 10-14 psi, with a 1-sec injection time. It is probably best to adjust the ejection pressure and keep the time constant, as oocytes do not tolerate extremely rapid injection. Incubation of Oocytes After the oocytes have been defolliculated or injected they should be maintained in a relatively isoosmotic solution containing calcium. A wide variety of solutions have been used for this purpose, including Barth's, 4 OR2 with calcium,~S normal frog Ringer's solution,19 L-15 medium, 2° and ts R. A. Wallace, D. W. Jared, J. N. Dumont, and M. W. Sega, J. Exp. Zool. 184, 321 (1973).
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ND962~ (recipes are given in Appendix I). ND96 is essentially OR2 which is slightly hypertonic (sodium concentration 96 compared to 82.5 mM), which may be beneficial when injecting the oocytes. In addition, the incubation medium should be supplemented with sodium pyruvate (550 mg/liter) as a carbon source22 and gentamicin (100 #g/ml) to prevent bacterial contamination.23 An alternative antibiotic mix that is effective is a combination of 100 U/ml penicillin and 100 #g/ml streptomycin.24 Theophylline can be added to a concentration of 0.5 m M to inhibit phosphodiesterase and keep cAMP level high, which will prevent maturation. This is generally not necessary, however, because cAMP levels are normally high in oocytes.25 The oocytes should be incubated in 35-mm diameter tissue culture dishes (60-ram diameter dishes for large numbers) on a rotator at slow speed in a 20 ° controlled temperature incubator. Temperature fluctuations, particularly heat, can both be damaging to oocyte viability and result in erratic time courses of expression. The healthy ooctyes should be transferred to fresh dishes with new ND96 each day, and more often if there is any oocyte death. This is particularly important shortly after injection, as dying oocytes release salts which make the solution hypertonic and toxic to the remainder. The time course of expression varies with the type of RNA injected. Detectable expression of sodium channels can be observed 24 hr after injection of RNA made in vitro from a clone when the oocytes are incubated at 20 ° (lower temperatures result in a longer delay). Expression of this channel reaches maximal levels by about 2 days and does not noticeably decrease at least until 6 days postinjection. Appendix I: Recipes OR2--calcium-free 82.5 mM NaC1 2mM KCI 1 mM MgC12 5 mM HEPES,pH 7.5 with NaOH
19C. Methfessel,V. Witzemann,T. Takahashi, M. Mishina, S. Numa, and B. Sakmann, Pfluegers Arch. 407, 577 (1986). 2oR. A. Wallace,Z. Misulovin,D. W. Jared, and H. S. Wiley,GameteRes. 1, 269 (1978). 21j. p. Leonard,J. Nargeot,T. P. Snutch,N. Davidson,and H. A. Lester,J. Neurosci, 7, 875 (1987). 22j. j. Eppigand J. N. Dumont,In Vitro 12, 418 (1976). 23R. A. Wallaceand Z. Misulovin,Proc. Natl. Acad. Sci. U.S.A. 75, 5534 (1978). 24R. A. Laskey,J. CellSci. 7, 653 (1970). 25M. F. Cicirelliand L. D. Smith,Dev. Biol. 108, 254 (1985).
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EXPRESSION OF ION CHANNELS
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ND96 96 mM NaCI 2 mM KC1 1.8 mM CaCl2 1 mM MgC12 5 mM HEPES, pH 7.5 with NaOH Modified Barth's solution 88 mM NaCl 1 mM KC1 2.4 mM NaHCO3 20 mM HEPES, pH 7.5 0.82 mM MgSO4 0.33 mM Ca(NO3)2 0.41 mM CaCI2 Normal frog Ringer's solution 115 mM NaC1 2.5 mMKC1 1.8 mM CaC12 10 mM HEPES, pH 7.2 L-15 70% Leibovitz's L-15 medium (available from GIBCO, Grand Island, NY) 10 mMHEPES, pH 7.5 100X Injection Dye 0.2 M Tris-HCl, pH 7.5 1 M NaC1 4% Trypan blue (Use 10× for needle calibration and IX for DNA samples)
Appendix II: Suppliers ofXenopus laevis United States Suppliers NASCO, 901 Janesville Ave., Fort Atkinson, WI 53538, (414) 563-2446. Ordering Information: LM535MP, adult females, 9-10.5 cm @ $20.60; LM531P, guaranteed ooeyte-positive females; LM535WC, adult females, 9-10.5 cm @ $17.20 (wild-caught); LM531WC, guaranteed oocyte-positivefemales (wild-caught). Notes: The first two catalog numbers are for laboratory-reared frogs. The last two are for wild-caught, laboratory-conditioned frogs (a recent addition to the catalog). The 9-10.5 cm adult females are generally the best source of mature oocytes, as these frogs are guaranteed to be large and have always been ooeyte positive. Xenopus I, 716 Northside, Ann Arbor, MI 48105, (313) 426-2083. Ordering Information: 4216, adult females, ooeyte positive, @ $13.90. Notes: Frogs captured in the wild. Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215, (800) 334-5551, (919) 584-0381. Ordering Information: L1570, adult females @ $18.79. Notes: Laboratory-reared frogs.
[ 16]
RNA PREPARATIONFOR INJECTION
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European Suppliers Xenopus Ltd., Holmesdaie Nursery, Mid Street, South Nutfield, RedhiU, Surrey RH1 4JY, England, 44-73-782-2687. Ordering Information: Very large, mature females. Notes: Either laboratory-reared or wild frogs. Dipl. Biol.-Dipl. Ing. Horst KAMer Institut f'tir Entwicklungsbiologle Kollaustrasse 113b D-2000 Hamburg 61 Germany 40-587675 Ordering Information: X. laevisor X. laevisalbinos Notes: Either laboratory-reared or wild frogs.
Japanese Suppliers Nippon Life Science, ordering address: 1-1-32 Shiba-Daimon, Minatoku, Tokyo 105; company address: Denmacho 19, Hamamatsu City, Shizuoka, Japan. Seibu Department Stores Tokyo, Japan Notes: Xenopus can be purchased in the departmem stores in Japan.
South African Supplier South African Snake Farm, P.O. Box 6, Fish Hock, Cape Province, Republic of South Africa. Notes: Frogs can be ordered directly from South Africa for air shipment anywhere in the world.
Acknowledgments The author is a LuciUe P. Markey Scholar, and work in his laboratory is supported by grants from the U.S. National Institutes of Health (NS-26729), the Lucille P. Markey Charitable Trust, and the March of Dimes Basil O'Connor Starter Scholar Program.
[ 16] Preparation of RNA for Injection into Xenopus Oocytes By ALAN L. GOLDIN a n d KATUMI SUMIKAWA Introduction
Xenopus oocytes are n o w a p o p u l a r system for the expression a n d characterization o f functional n e u r o t r a n s m i t t e r receptors a n d ion channels. O o c y t e s are injected with m R N A s extracted f r o m tissues or syntheMETHODS IN ENZYMOLOGY, VOL. 207
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