- Email: [email protected]
[28] Microinjection into Xenopus Oocytes: Equipment
276
PREPARATION AND CHARACTERIZATIONOF RNA
[28]
ing 2 mM EGTA and centrifuged at 100,000 g for 30 min at 4°. The resulting pellet is resuspended in 0.25 M sucrose in TK to 50 A260units/ml and stored in liquid nitrogen. Alternatives to Membrane Preparations. If microsomes are only used to maximize translation, it may be possible to substitute 0.1-0.5% (v/v) T r i t o n X - I 0 0 . 7 The mechanism by which detergents exert their stimulatory effect and substitute for microsomes is poorly understood, but implies that translation is inhibited in the absence of membranes by some sort of hydrophobic interaction. Cautions in the Use of Microsomes. Although much data in the literature suggest that the signals and components involved in the translation and processing of membrane, secretory, and lysosomal proteins are universal, significant differences have been noted in the responses of the reticulocyte and wheat germ translation systems to the addition of microsomes and more highly purified components.~° The investigator studying in vitro translation of mRNAs coding for such proteins may have to find out what works for him.
[28] M i c r o i n j e c t i o n into X e n o p u s O o c y t e s : E q u i p m e n t B y MICHAEL J. M. HITCHCOCK, EDWARD I. GINNS, and CAROL J. MARCUS-SEKURA
The microinjection of nucleic acids into Xenopus oocytes requires accurate delivery of volumes in the nanoliter range. The capablity of performing injections from a single filling of the needle facilitates processing of large numbers of samples. This chapter describes the construction and operation of a microinjection device fulfilling these criteria. The reproducibility of the apparatus depends on the fluid transmission of a mechanical impulse using bubble-free oil. A glass microinjection needle (Figs. Ib and 2b) is attached to the tip of a syringe needle which is completely filled with oil (Fig. 2b). Movement of the syringe plunger is controlled by a stepping motor (Fig. 2b), and the oil provides positive displacement for moving the sample fluid in the glass injection needle (Fig. 2b). The sample only makes contact with the glass injection needle and the oil. The stepping motor is mounted directly onto a micromanipulator (Fig. Ic) in order to eliminate the need for flexible tubing connections between the injection needle and the fluid drive. i M. J. M. Hitchcock and R. M. Friedman, Anal. Biochem. 109, 338 (1980).
METHODS IN ENZYMOLOGY, VOL. 152
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[28]
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Construction of the Microinjector The microinjector is composed of two main parts: a micromanipulator (Fig. la, part B) and a microprocessor-controlled pipettor (Fig. 1, b and c). Fluid delivery is accomplished with a Micro Lab P programmable microprocessor-controlled pipettor (Fig. lc; Hamilton, Reno, NV) equipped with an external speed controller (Fig. lc, part H) and a foot-operated switch. A MM33 micromanipulator (Fig. lc, part F) without the fine thrust drive and spacer (Brinkmann Instruments, Inc., Westbury, NY) controls movement of the needle. The coarse thrust drive of the micromanipulator is disconnected and positioned on the solid half of the case as shown in Fig. la. For assembly of the device, the two halves of the plastic case of the hand pipettor of the Micro Lab P are disassembled. The positions of the screw holes are marked and after the holes are drilled in the plastic, the two pieces (coarse thrust drive and half of the plastic case) are bolted together (Fig. la). The hand pipettor is then reassembled and the thrust drive is reattached to the micromanipulator (Fig. lb). The micromanipulator holds the needle at an oblique angle to a microscope stage for injection (Fig. lc). The manipulator controls permit the smooth vertical and horizontal movements needed to fill the needle and inject oocytes. The pipettor is fitted with a Hamilton 7000 series syringe (7105N, 5 ~1; or 7107N, 1 tzl) using the 1705-1750 adapter supplied with the Micro Lab P (Fig. lb, part C). The plunger buttons of these syringes are dome shaped and must be filed flat for them to fit. Polyethylene tubing (0.58 mm i.d., 0.965 mm o.d.) is used to cover all except 6 mm of the wire needle tip of the syringe (Fig. 2b). A 14-gauge cannula (4 in. original length, Becton Dickinson Co., Rutherford, N J) lined with another piece of polyethylene tubing (I. 14 mm i.d., 1.57 mm o.d.) and cut to leave about 1 cm of the wire needle exposed is fitted over this tubing and wire needle to provide more rigidity (Fig. 2). During the cutting of the cannula, care must be taken to avoid burrs and distortion of the cannula. Two centimeters of silicon tubing (0.79 mm i.d., 3.18 mm o.d.) is pushed over the end of the cannula to cover the needle, polyethylene tubing, and tip of the cannula, leaving about 1 mm of the wire needle exposed (see Figure 2b). The hand pipettor attached to the micromanipulator is then mounted on a stand, the foot-controlled switch is connected to the external speed controller, and the leads from the hand pipettor and external speed controller are connected to the main control unit (Fig. Ic). Microinjection needles (3.5-4.0 cm long) are prepared by pulling out glass capillaries (20 ~1; Microcaps, Drummond Scientific Co., Broomall, PA) to approximately 25/zm outside diameter using a micropipet puller
278
PREPARATION AND CHARACTERIZATION OF R N A
b
[28]
B
Fro. 1. The microinjection apparatus. (a) Approximate locations of the holes that are drilled in one side of the plastic case of the pipettor (part A) so that they align with the corresponding holes in the coarse thrust drive of the micromanipulator (part B). (b) View of the reassembled hand pipettor (part A) with the micromanipulator drive (part B) bolted in place. A syringe (part C) is fitted with a needle covered by a cannula (part D). The boxed area (labeled E) around the needle tip is shown diagrammatically in more detail in Fig. 2.
MICROINJECTION INTOXenopus OOCYTES
[28]
279 a
GLASS INJECTIONNEEDLE
CANNt.ILA
,,L,0o.,,v.,.0
SYRINGEBARREL
I--o.6omq
"1
"l
b
,r SYRINGEN E E D L E
POLYETHYLENE TUBING~
FIG. 2. (a) Schematic representation of oocyte microinjection apparatus showing syringe-injection needle assembly. (b) Expanded view of the glass injection needle assembly.
(J. B. Kefe, Islington, MA). The needles are conveniently stored point up in a piece of styrofoam and placed in a plastic container to prevent contamination and injury. Operation of the Microinjection Apparatus
Fitting the Glass Needle 1. The syringe must be empty (see steps 1-5 of the following two sections). 2. Break off the old glass needle tip and then remove the remaining glass barrel of the old needle. Removing the old glass needle without breaking off the tip will introduce bubbles into the syringe needle. 3. Hold a new glass needle against a contrasting background and with ethanol-flamed tweezers touch the tip to open the seal. 4. A 3-6 ml syringe is filled with paraffin oil and fitted with a 0.5-in. 24-gauge needle. Bubbles are removed from the needle of this syringe, (c) Illustration of the microinjection apparatus during injections. The micromanipulator thrust drive (part B) has been reattached to the rest of the micromanipulator (part F) and then mounted on a ring stand. The wire for the foot-operated switch (part G) is connected to the external speed controller (part H) which is taped onto the microprocessor control unit (part I).
280
PREPARATION AND CHARACTERIZATION OF R N A
[28]
and the syringe needle is inserted into the wide end of the glass needle to fill it completely with oil. 5. After filling the glass needle with oil, maintain pressure on the filling syringe barrel while separating the needles in order to prevent air bubbles from forming. 6. Attach the glass needle to the pipettor by sliding it inside the silicon tubing until it rests over the wire of the Hamilton syringe (Fig. 2b). Too much pressure will cause air to appear in the glass needle.
Clearing Bubbles from the Glass Needle 1. Switch on the Micro lab P [the C button on the speed controller is in the off (out) position]. 2. Enter # (syringe volume in nanoliters), E. 3. Press *, SPEED, 1. (Display shows E for external.) 4. Press *, MAN. 5. Press START twice. 6. Fit needle (if not already on). 7. Turn the pipettor vertically so that needle points up. 8. Set speed controller at slowest speed (Fig. lc, part H). 9. Press C button on speed controller (syringe plunger withdraws for approximately 3 min). 10. Release C button. 11. Set speed controller to fastest speed. 12. Press C on speed controller until syringe plunger returns (approximately 3 sec). 13. Release C button. If bubbles are visible in the needle tip, repeat steps 8-13 until no bubbles are produced during three cycles of the above procedure.
Injection Procedure 1. Switch on Micro Lab P main controller [C button on the speed controller must be in the off (out) position]. 2. Enter # (syringe volume in nanoliters), E. Press *, SPEED, 1. (Display shows E for external.) 4. Press *, RDIS, # (injection volume in nl), E, # (number of injections + 2), E. Note the volume shown in the volume display. 5. Press START twice. 6. Place a sterile plastic plate on the microscope stage (Falcon lid 3041) and place a sterile glass microscope slide on it (Figs. lc and 3). 7. Set the speed controller to 40 nl/sec. .
[28]
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281
8. Fit a glass injection needle (see previous section) if needed. 9. Line up the needle at an angle sloping toward the stage (Figs. lc and 3) and within the microscope field of view. 10. Maneuver the needle straight up, without changing its angle to the stage, with the vertical micromanipulator control. 11. Place the sample of DNA or RNA to be injected as a droplet on the plastic plate (use 500 nl or the total volume programmed in step 4 plus 200 nl, whichever is larger). 12. While viewing the sample droplet through the microscope bring the needle, still angled at the stage, to the center of the sample droplet, close to, or just touching the plastic lid. 13. Press foot pedal (same as START) to fill the syringe with the sample. 14. After filling is complete, change the speed controller setting to approximately 100 nl/sec. 15. Press foot pedal (or START) to dispense one injection aliquot. 16. Place the oocytes to be injected at the center of the plastic lid in a small amount of modified Barth's medium. The oocytes must not dry out during the injection procedure. The number of oocytes injected at a time is determined by the experience of the operator--initially 5, later 20. 17. Slide the glass microscope slide toward the oocytes until the media is pulled back under the slide. Using blunt, sterile forceps, position the oocytes along the edge of the slide with the dark animal poles toward the needle (see Fig. 3). Although we suggest injection into the dark pole, some investigators inject into the light vegetal pole, while others inject at
POSITIONOF FOREFINGER GLASS NEEDLE OOCYTE
I
GLASS SLIDE
l MEDIUM
PLASTIC TRAY
FIG. 3. Diagram illustrating the microinjection of mRNA into oocyte cytoplasm.
282
PREPARATION AND CHARACTERIZATION OF R N A
[28]
the equator. Injection at any of these sites results in translation of mRNA, but regardless of the site chosen one must avoid damage to the nucleus. 18. Move the plastic plate, holding the slide in place, such that the first oocyte is at the bottom of the field of view. 19. Bring the needle, still angled to the stage, into the field of view and adjust its position so that it will pierce the dark pole of the oocyte at 2030°. 20. Insert the needle into the dark pole of oocyte using the micromanipulator controls. 21. Inject the sample by pressing the foot pedal (or START). If the injected volume is 20 nl or greater, the oocyte should transiently swell. 22. Remove the needle from the oocyte using the micromanipulator. 23. Move the needle to the next oocyte. 24. Continue injection by repeating steps 21-24. 25. When all oocytes have been injected, raise the needle using the micromanipulator controls. 26. Lift the glass slide off the plastic lid and pipet medium onto the oocytes. With a wide-mouth Pasteur pipet transfer the injected oocytes into modified Barth's medium. 27. Repeat steps 17-26 until the number of steps indicated on the Micro Lab P main controller display is 1. This should coincide with the final oocyte injection. 28. Bring the injection needle down to the plastic lid and press start to remove the final aliquot of the sample. If the operation has been carried out successfully, the oil-water interface can be seen moving to the tip of the needle. A small oil bubble is often seen in the aqueous droplet on the plastic lid. 29. Repeat steps 7-28 for subsequent injections of samples of the same volume using the same number of oocytes, or steps 4-28 (step 6 can be omitted) if the volume and/or the number are to be changed. 30. See next section for shutdown.
Storage of Microinjection Apparatus 1. After the final injection, proceed through steps 4-10 of the second section, Clearing Bubbles from the Glass Needle. 2. Switch off the main controller, leaving the syringe filled with distilled water. Bubble formation is kept to a minimum by keeping the injector tip up. The microinjection apparatus can be prepared for use by following steps 1-5 of the previous section, Injection Procedure, using the fastest setting on the speed controller.
[28]
MICROINJECTION INTO
Xenopus OOCYTES
283
Microinjection o f DNA into the Germinal Vesicle The micropipettor assembly described above can readily be adapted for microinjection of DNA into the oocyte germinal vesicle (nucleus). As described in the next chapter [29], oocytes are prepared so that the germinal vesicle is floating at the top surface of the oocyte. The oocytes are positioned on a Spectramesh grid in a small plastic petri dish (Fig. 4). Injection into the nucleus required a vertical angle of injection rather than the oblique angle used for cytoplasmic injections. To accomplish this, the Hamilton pipettor is positioned at the same angle used for cytoplasmic injections, but the glass capillary needle is modified (Fig. 4). Capillaries to be used for these needles are initially pulled with a slightly longer length of barrel tubing (approximately 4.5 cm) than those needles used for cytoplasmic injection. The glass needle tip is broken off by touching it gently with a pair of sterile forceps. Then, while the needle is held with a pair of forceps on the needle barrel close to the tip, the midpoint of the needle is briefly positioned horizontally in a Bunsen burner flame. When the end of the needle away from the tip bends to form an angle of approximately 120°, the needle is pulled out of the flame. After cooling, the needle is filled with paraffin oil and inserted onto the Hamilton syringe as described in Fitting the Glass Needle. The addition of a small amount of an oilsoluble dye, such as O-cresol red to the paraffin oil, makes it easier to visualize the needle in the subsequent microinjection manipulations. Plasmid DNA at a concentration of 1 mg/ml has been successfully
GLASS
~ P OOCYTES=""'-~
E
~
T
R
I
DISH
SPECTRAMESH GRID FIG.4. Diagramillustratingthemicroinjectionof DNAintothegerminalvesiclesof oocytcs.
284
PREPARATION AND CHARACTERIZATION OF RNA
[29]
used in protein expression experiments. 2 The DNA should be centrifuged in a microfuge for 2 min immediately prior to loading into the needle to precipitate particulates which might block the needle. After centrifugation, an aliquot is removed from the DNA solution (avoiding any pellet), placed onto a plastic lid, and an amount sufficient to inject 20-30 oocytes with 15 nl each is loaded into the needle (as described in the Injection Procedures). It is preferable to inject an entire batch of prepared oocytes rapidly because oocyte debris collects on the tip of the needle and, when dry, blocks the needle. Injection of 50 oocytes per DNA sample should provide a detectable level of protein expression. 2 R. J. Watson, A. M. Colberg-Poley, C. J. Marcus-Sekura, B. J. Carter, and L. W. Enquist,
Nucleic Acids Res. 11, 1507 (1983).
[29] P r e p a r a t i o n o f O o c y t e s for M i c r o i n j e c t i o n o f R N A and DNA B y CAROL J. M A R C U S - S E K U R A a n d M I C H A E L J. M . HITCHCOCK
The oocytes of the African clawed toad, Xenopus laevis, have been used extensively to test the biological activity of purified macromolecules. The Xenopus oocyte provides a unique unicellular test system for the study of transcription,l-3 translation,4,5 and secretion6 of injected molecules. The oocyte will posttranscriptionally process proteins it synthesizes from injected DNA or RNA and will also secrete those proteins normally secreted in vivo. This chapter will describe a protocol by which oocytes are surgically removed and prepared for microinjection of RNA into the oocyte cytoplasm or DNA into the oocyte nucleus (germinal J. E. Mertz and J. B. Gurdon, Proc. Natl. Acad. Sci. U.S.A. 74, 1502 (1977). 2 S. L. McKnight, E. R. Garvis, R. Kingsbury, and R. Axel, Cell (Cambridge, Mass.) 25, 385 (1981). 3 E. M. de Robertis and J. E. Mertz, Cell (Cambridge, Mass.) 12, 175 (1977). 4 j. B. Gurdon, C. D. Lane, H. R. Woodland, and G. Marbaix, Nature (London) 233, 177 (1971). 5 S. L. Berger, M. J. M. Hitchcock, K. C. Zoon, C. S. Birkenmeier, R. M. Friedman, and E. H. Chang, J. Biol. Chem. 255, 2955 (1980). 6 A. Colman, C. D. Lane, R. Craig, A. Boulton, T. Mohun, and J. Morser, Fur. J. Biochem. 113, 339 (1981).
METHODS IN ENZYMOLOGY, VOL. 152
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
PREPARATION AND CHARACTERIZATIONOF RNA
[28]
ing 2 mM EGTA and centrifuged at 100,000 g for 30 min at 4°. The resulting pellet is resuspended in 0.25 M sucrose in TK to 50 A260units/ml and stored in liquid nitrogen. Alternatives to Membrane Preparations. If microsomes are only used to maximize translation, it may be possible to substitute 0.1-0.5% (v/v) T r i t o n X - I 0 0 . 7 The mechanism by which detergents exert their stimulatory effect and substitute for microsomes is poorly understood, but implies that translation is inhibited in the absence of membranes by some sort of hydrophobic interaction. Cautions in the Use of Microsomes. Although much data in the literature suggest that the signals and components involved in the translation and processing of membrane, secretory, and lysosomal proteins are universal, significant differences have been noted in the responses of the reticulocyte and wheat germ translation systems to the addition of microsomes and more highly purified components.~° The investigator studying in vitro translation of mRNAs coding for such proteins may have to find out what works for him.
[28] M i c r o i n j e c t i o n into X e n o p u s O o c y t e s : E q u i p m e n t B y MICHAEL J. M. HITCHCOCK, EDWARD I. GINNS, and CAROL J. MARCUS-SEKURA
The microinjection of nucleic acids into Xenopus oocytes requires accurate delivery of volumes in the nanoliter range. The capablity of performing injections from a single filling of the needle facilitates processing of large numbers of samples. This chapter describes the construction and operation of a microinjection device fulfilling these criteria. The reproducibility of the apparatus depends on the fluid transmission of a mechanical impulse using bubble-free oil. A glass microinjection needle (Figs. Ib and 2b) is attached to the tip of a syringe needle which is completely filled with oil (Fig. 2b). Movement of the syringe plunger is controlled by a stepping motor (Fig. 2b), and the oil provides positive displacement for moving the sample fluid in the glass injection needle (Fig. 2b). The sample only makes contact with the glass injection needle and the oil. The stepping motor is mounted directly onto a micromanipulator (Fig. Ic) in order to eliminate the need for flexible tubing connections between the injection needle and the fluid drive. i M. J. M. Hitchcock and R. M. Friedman, Anal. Biochem. 109, 338 (1980).
METHODS IN ENZYMOLOGY, VOL. 152
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
[28]
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277
Construction of the Microinjector The microinjector is composed of two main parts: a micromanipulator (Fig. la, part B) and a microprocessor-controlled pipettor (Fig. 1, b and c). Fluid delivery is accomplished with a Micro Lab P programmable microprocessor-controlled pipettor (Fig. lc; Hamilton, Reno, NV) equipped with an external speed controller (Fig. lc, part H) and a foot-operated switch. A MM33 micromanipulator (Fig. lc, part F) without the fine thrust drive and spacer (Brinkmann Instruments, Inc., Westbury, NY) controls movement of the needle. The coarse thrust drive of the micromanipulator is disconnected and positioned on the solid half of the case as shown in Fig. la. For assembly of the device, the two halves of the plastic case of the hand pipettor of the Micro Lab P are disassembled. The positions of the screw holes are marked and after the holes are drilled in the plastic, the two pieces (coarse thrust drive and half of the plastic case) are bolted together (Fig. la). The hand pipettor is then reassembled and the thrust drive is reattached to the micromanipulator (Fig. lb). The micromanipulator holds the needle at an oblique angle to a microscope stage for injection (Fig. lc). The manipulator controls permit the smooth vertical and horizontal movements needed to fill the needle and inject oocytes. The pipettor is fitted with a Hamilton 7000 series syringe (7105N, 5 ~1; or 7107N, 1 tzl) using the 1705-1750 adapter supplied with the Micro Lab P (Fig. lb, part C). The plunger buttons of these syringes are dome shaped and must be filed flat for them to fit. Polyethylene tubing (0.58 mm i.d., 0.965 mm o.d.) is used to cover all except 6 mm of the wire needle tip of the syringe (Fig. 2b). A 14-gauge cannula (4 in. original length, Becton Dickinson Co., Rutherford, N J) lined with another piece of polyethylene tubing (I. 14 mm i.d., 1.57 mm o.d.) and cut to leave about 1 cm of the wire needle exposed is fitted over this tubing and wire needle to provide more rigidity (Fig. 2). During the cutting of the cannula, care must be taken to avoid burrs and distortion of the cannula. Two centimeters of silicon tubing (0.79 mm i.d., 3.18 mm o.d.) is pushed over the end of the cannula to cover the needle, polyethylene tubing, and tip of the cannula, leaving about 1 mm of the wire needle exposed (see Figure 2b). The hand pipettor attached to the micromanipulator is then mounted on a stand, the foot-controlled switch is connected to the external speed controller, and the leads from the hand pipettor and external speed controller are connected to the main control unit (Fig. Ic). Microinjection needles (3.5-4.0 cm long) are prepared by pulling out glass capillaries (20 ~1; Microcaps, Drummond Scientific Co., Broomall, PA) to approximately 25/zm outside diameter using a micropipet puller
278
PREPARATION AND CHARACTERIZATION OF R N A
b
[28]
B
Fro. 1. The microinjection apparatus. (a) Approximate locations of the holes that are drilled in one side of the plastic case of the pipettor (part A) so that they align with the corresponding holes in the coarse thrust drive of the micromanipulator (part B). (b) View of the reassembled hand pipettor (part A) with the micromanipulator drive (part B) bolted in place. A syringe (part C) is fitted with a needle covered by a cannula (part D). The boxed area (labeled E) around the needle tip is shown diagrammatically in more detail in Fig. 2.
MICROINJECTION INTOXenopus OOCYTES
[28]
279 a
GLASS INJECTIONNEEDLE
CANNt.ILA
,,L,0o.,,v.,.0
SYRINGEBARREL
I--o.6omq
"1
"l
b
,r SYRINGEN E E D L E
POLYETHYLENE TUBING~
FIG. 2. (a) Schematic representation of oocyte microinjection apparatus showing syringe-injection needle assembly. (b) Expanded view of the glass injection needle assembly.
(J. B. Kefe, Islington, MA). The needles are conveniently stored point up in a piece of styrofoam and placed in a plastic container to prevent contamination and injury. Operation of the Microinjection Apparatus
Fitting the Glass Needle 1. The syringe must be empty (see steps 1-5 of the following two sections). 2. Break off the old glass needle tip and then remove the remaining glass barrel of the old needle. Removing the old glass needle without breaking off the tip will introduce bubbles into the syringe needle. 3. Hold a new glass needle against a contrasting background and with ethanol-flamed tweezers touch the tip to open the seal. 4. A 3-6 ml syringe is filled with paraffin oil and fitted with a 0.5-in. 24-gauge needle. Bubbles are removed from the needle of this syringe, (c) Illustration of the microinjection apparatus during injections. The micromanipulator thrust drive (part B) has been reattached to the rest of the micromanipulator (part F) and then mounted on a ring stand. The wire for the foot-operated switch (part G) is connected to the external speed controller (part H) which is taped onto the microprocessor control unit (part I).
280
PREPARATION AND CHARACTERIZATION OF R N A
[28]
and the syringe needle is inserted into the wide end of the glass needle to fill it completely with oil. 5. After filling the glass needle with oil, maintain pressure on the filling syringe barrel while separating the needles in order to prevent air bubbles from forming. 6. Attach the glass needle to the pipettor by sliding it inside the silicon tubing until it rests over the wire of the Hamilton syringe (Fig. 2b). Too much pressure will cause air to appear in the glass needle.
Clearing Bubbles from the Glass Needle 1. Switch on the Micro lab P [the C button on the speed controller is in the off (out) position]. 2. Enter # (syringe volume in nanoliters), E. 3. Press *, SPEED, 1. (Display shows E for external.) 4. Press *, MAN. 5. Press START twice. 6. Fit needle (if not already on). 7. Turn the pipettor vertically so that needle points up. 8. Set speed controller at slowest speed (Fig. lc, part H). 9. Press C button on speed controller (syringe plunger withdraws for approximately 3 min). 10. Release C button. 11. Set speed controller to fastest speed. 12. Press C on speed controller until syringe plunger returns (approximately 3 sec). 13. Release C button. If bubbles are visible in the needle tip, repeat steps 8-13 until no bubbles are produced during three cycles of the above procedure.
Injection Procedure 1. Switch on Micro Lab P main controller [C button on the speed controller must be in the off (out) position]. 2. Enter # (syringe volume in nanoliters), E. Press *, SPEED, 1. (Display shows E for external.) 4. Press *, RDIS, # (injection volume in nl), E, # (number of injections + 2), E. Note the volume shown in the volume display. 5. Press START twice. 6. Place a sterile plastic plate on the microscope stage (Falcon lid 3041) and place a sterile glass microscope slide on it (Figs. lc and 3). 7. Set the speed controller to 40 nl/sec. .
[28]
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8. Fit a glass injection needle (see previous section) if needed. 9. Line up the needle at an angle sloping toward the stage (Figs. lc and 3) and within the microscope field of view. 10. Maneuver the needle straight up, without changing its angle to the stage, with the vertical micromanipulator control. 11. Place the sample of DNA or RNA to be injected as a droplet on the plastic plate (use 500 nl or the total volume programmed in step 4 plus 200 nl, whichever is larger). 12. While viewing the sample droplet through the microscope bring the needle, still angled at the stage, to the center of the sample droplet, close to, or just touching the plastic lid. 13. Press foot pedal (same as START) to fill the syringe with the sample. 14. After filling is complete, change the speed controller setting to approximately 100 nl/sec. 15. Press foot pedal (or START) to dispense one injection aliquot. 16. Place the oocytes to be injected at the center of the plastic lid in a small amount of modified Barth's medium. The oocytes must not dry out during the injection procedure. The number of oocytes injected at a time is determined by the experience of the operator--initially 5, later 20. 17. Slide the glass microscope slide toward the oocytes until the media is pulled back under the slide. Using blunt, sterile forceps, position the oocytes along the edge of the slide with the dark animal poles toward the needle (see Fig. 3). Although we suggest injection into the dark pole, some investigators inject into the light vegetal pole, while others inject at
POSITIONOF FOREFINGER GLASS NEEDLE OOCYTE
I
GLASS SLIDE
l MEDIUM
PLASTIC TRAY
FIG. 3. Diagram illustrating the microinjection of mRNA into oocyte cytoplasm.
282
PREPARATION AND CHARACTERIZATION OF R N A
[28]
the equator. Injection at any of these sites results in translation of mRNA, but regardless of the site chosen one must avoid damage to the nucleus. 18. Move the plastic plate, holding the slide in place, such that the first oocyte is at the bottom of the field of view. 19. Bring the needle, still angled to the stage, into the field of view and adjust its position so that it will pierce the dark pole of the oocyte at 2030°. 20. Insert the needle into the dark pole of oocyte using the micromanipulator controls. 21. Inject the sample by pressing the foot pedal (or START). If the injected volume is 20 nl or greater, the oocyte should transiently swell. 22. Remove the needle from the oocyte using the micromanipulator. 23. Move the needle to the next oocyte. 24. Continue injection by repeating steps 21-24. 25. When all oocytes have been injected, raise the needle using the micromanipulator controls. 26. Lift the glass slide off the plastic lid and pipet medium onto the oocytes. With a wide-mouth Pasteur pipet transfer the injected oocytes into modified Barth's medium. 27. Repeat steps 17-26 until the number of steps indicated on the Micro Lab P main controller display is 1. This should coincide with the final oocyte injection. 28. Bring the injection needle down to the plastic lid and press start to remove the final aliquot of the sample. If the operation has been carried out successfully, the oil-water interface can be seen moving to the tip of the needle. A small oil bubble is often seen in the aqueous droplet on the plastic lid. 29. Repeat steps 7-28 for subsequent injections of samples of the same volume using the same number of oocytes, or steps 4-28 (step 6 can be omitted) if the volume and/or the number are to be changed. 30. See next section for shutdown.
Storage of Microinjection Apparatus 1. After the final injection, proceed through steps 4-10 of the second section, Clearing Bubbles from the Glass Needle. 2. Switch off the main controller, leaving the syringe filled with distilled water. Bubble formation is kept to a minimum by keeping the injector tip up. The microinjection apparatus can be prepared for use by following steps 1-5 of the previous section, Injection Procedure, using the fastest setting on the speed controller.
[28]
MICROINJECTION INTO
Xenopus OOCYTES
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Microinjection o f DNA into the Germinal Vesicle The micropipettor assembly described above can readily be adapted for microinjection of DNA into the oocyte germinal vesicle (nucleus). As described in the next chapter [29], oocytes are prepared so that the germinal vesicle is floating at the top surface of the oocyte. The oocytes are positioned on a Spectramesh grid in a small plastic petri dish (Fig. 4). Injection into the nucleus required a vertical angle of injection rather than the oblique angle used for cytoplasmic injections. To accomplish this, the Hamilton pipettor is positioned at the same angle used for cytoplasmic injections, but the glass capillary needle is modified (Fig. 4). Capillaries to be used for these needles are initially pulled with a slightly longer length of barrel tubing (approximately 4.5 cm) than those needles used for cytoplasmic injection. The glass needle tip is broken off by touching it gently with a pair of sterile forceps. Then, while the needle is held with a pair of forceps on the needle barrel close to the tip, the midpoint of the needle is briefly positioned horizontally in a Bunsen burner flame. When the end of the needle away from the tip bends to form an angle of approximately 120°, the needle is pulled out of the flame. After cooling, the needle is filled with paraffin oil and inserted onto the Hamilton syringe as described in Fitting the Glass Needle. The addition of a small amount of an oilsoluble dye, such as O-cresol red to the paraffin oil, makes it easier to visualize the needle in the subsequent microinjection manipulations. Plasmid DNA at a concentration of 1 mg/ml has been successfully
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PREPARATION AND CHARACTERIZATION OF RNA
[29]
used in protein expression experiments. 2 The DNA should be centrifuged in a microfuge for 2 min immediately prior to loading into the needle to precipitate particulates which might block the needle. After centrifugation, an aliquot is removed from the DNA solution (avoiding any pellet), placed onto a plastic lid, and an amount sufficient to inject 20-30 oocytes with 15 nl each is loaded into the needle (as described in the Injection Procedures). It is preferable to inject an entire batch of prepared oocytes rapidly because oocyte debris collects on the tip of the needle and, when dry, blocks the needle. Injection of 50 oocytes per DNA sample should provide a detectable level of protein expression. 2 R. J. Watson, A. M. Colberg-Poley, C. J. Marcus-Sekura, B. J. Carter, and L. W. Enquist,
Nucleic Acids Res. 11, 1507 (1983).
[29] P r e p a r a t i o n o f O o c y t e s for M i c r o i n j e c t i o n o f R N A and DNA B y CAROL J. M A R C U S - S E K U R A a n d M I C H A E L J. M . HITCHCOCK
The oocytes of the African clawed toad, Xenopus laevis, have been used extensively to test the biological activity of purified macromolecules. The Xenopus oocyte provides a unique unicellular test system for the study of transcription,l-3 translation,4,5 and secretion6 of injected molecules. The oocyte will posttranscriptionally process proteins it synthesizes from injected DNA or RNA and will also secrete those proteins normally secreted in vivo. This chapter will describe a protocol by which oocytes are surgically removed and prepared for microinjection of RNA into the oocyte cytoplasm or DNA into the oocyte nucleus (germinal J. E. Mertz and J. B. Gurdon, Proc. Natl. Acad. Sci. U.S.A. 74, 1502 (1977). 2 S. L. McKnight, E. R. Garvis, R. Kingsbury, and R. Axel, Cell (Cambridge, Mass.) 25, 385 (1981). 3 E. M. de Robertis and J. E. Mertz, Cell (Cambridge, Mass.) 12, 175 (1977). 4 j. B. Gurdon, C. D. Lane, H. R. Woodland, and G. Marbaix, Nature (London) 233, 177 (1971). 5 S. L. Berger, M. J. M. Hitchcock, K. C. Zoon, C. S. Birkenmeier, R. M. Friedman, and E. H. Chang, J. Biol. Chem. 255, 2955 (1980). 6 A. Colman, C. D. Lane, R. Craig, A. Boulton, T. Mohun, and J. Morser, Fur. J. Biochem. 113, 339 (1981).
METHODS IN ENZYMOLOGY, VOL. 152
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