Easy-to-use equipment for the accurate microinjection of nanoliter volumes into the nuclei of amphibian oocytes

Easy-to-Use

Equipment for the Accurate Microinjection of Nanoliter Volumes into the Nuclei of Amphibian Oocytes

DONALD

L. STEPHENS,* DAVID

*Departmem Wisrwwin

TIMOTHY ZIPsER,t AND

C/J’ 0ncolog.v. Mc,4rdlr Lahorutoq~ 53706. und tC‘old Spring Harbor Received

J. MILLER,* JANET

Jbr Cancer Laboratory. January

E.

LOUISE SII.VER,~.‘.

MERTZ,*.’

Research. Uniwrsity oJ’ Wisconsin. Cold Spring Harbor. !Yew York

,Madi.ron. I I724

5. 1981

Microinjection of Xwwpu.c oocytes has become a valuable technique for studying many aspects of control of eucaryotic gene expression. Previously published methods for controlling and monitoring the volume of sample injected into oocytes are time consuming and tedious and require considerable expertise to obtain reproducible results. The construction and manner of usage of micropipets are the most critical parts of a successful injection technique. Methods for the construction of micropipets and ways to assess the accuracy and precision of the injection of nanoliter volumes into amphibian oocytes are described. Construction of pipets is done almost entirely using instruments that are relatively inexpensive and allow for a high degree of reproducibility. Micropipets made in this manner have several advantages: (I ) They inflict little damage on the oocytes during the injection process: greater than 959 of the oocytes survive after microtnJection. (2) Oocytes do not require defolliculation prior to microinjection. (3) One micropipet can be used to inject as many as 100 oocytes before needing to be refilled with sample and without acquiring a blockage. Using the microinjection techniques described. the accuracy of sample delivery was within 9% of the mean and the precision increased with increasing volume: the standard deviations of volumes in the 5-nl range were wtthin 9% of the mean and volumes in the range IO-50 nl were within 4&h% of the mean.

Oocytes taken from the South African osteliunz discoideum ( 12) and a wide variety clawed frog .Yenopus laevis have been used of viral proteins ( 13.-16). in a wide variety of biological studies. FaithTranslation in .Yenopus oocytes has sevful translation of heterologous mRNA mi- eral advantages over conventional in virro croinjected into oocytes (1,2) has been used protein synthesizing systems. First, the into identify and quantify mRNA from many jected mRNAs continue to be translated for different sources. Translation in Xenopus long periods of time (24--48 h) ( I7), enabling oocytes of mRNAs from animal origins in- one to detect translational products of clude those coding for interferon (3.4). lens mRNAs that are only available in minute cystallins (5), uteroglobin (6), thyroglobin quantities. Second, Xenopzts oocytes per(7), protamine (8) proinsulin (9) and pro- form post-translational modifications of promellitin (IO). Plant mRNAs are also trans- teins in a manner similar to that done by the lated in Xenopus oocytes (I I), as well as cells from which the mRNAs were isolated mRNAs for secretory proteins from Dictv(7,8,1 1.13.14,18), thereby allowing one to assay for the synthesis of biologically active ’ Present address: Brookhaven National Laboratory, products. Third, when mRNAs coding for Upton. N. Y. 11973. exported proteins are injected into oocytes, ’ Further details concerning the construction and use the translational products are sequestered of the equipment described here can be obtained by writing to J.E.M’. at the above address. into vesicles(20.2 1) and transported ( 10.22).

300

STEPHENS

Xenopus oocytes can also be used to study transcription of eucaryotic DNAs microinjetted into the nucleus (23 25). The injected DNAs are reconstituted into chromatin (2628) (T. J. Miller, D. L. Stephens, and J. E. MertL, manuscript in preparation) and transcribed faithfully using the appropriate RNA polymerase ( 15,27). In addition, oocytes can correctly splice ( 15,16,29,30), modify (30), polyadenylate (3 1) (Miller et al., manuscript in preparation), transport (3 1,32) (Miller et al., manuscript in preparation), and, subsequently, translate ( 15,16,3 1) the initial transcripts. Thus, microinjection of Xenopus oocytes has proven to be a very useful technique for studying various aspects of control of eucaryotic gene expression. The published literature describing the technical aspects of the microinjection of amphibian oocytes is somewhat limited. The most widely cited paper (2) presents only a sketchy outline of the methodology. Other references (19,33,34) expand upon and illustrate it. Nevertheless, the approach to microinjection described in this publication entails several technical problems. (i) Each glass micropipet has to be calibrated separately. (ii) Movement of the fluid into the oocyte is controlled by a micrometer syringe coupled, via flexible tubing, to a micropipet. The tubing, usually filled with mineral oil, contains a compressible air space. Consequently, the micrometer syringe must be advanced to expel the fluid and retracted to stop the flow. Thus, yolk protein is pulled into the tip causing frequent clogging. (iii) One of the investigator’s hands is usually used to hold the oocytes in place, while the other is moved around to control the insertion of the micropipet, focusing of the microscope, and expulsion of the fluid. Therefore, the technique requires considerable expertise to obtain reproducible and accurate injections. This paper describes, in detail, techniques that were designed to enable investigators to inject oocytes successfully and reproducibly without being subject to the difficulties

ET AL

mentioned above. These difficulties were overcome by: (i) the construction of micropipets from uniform-bore tubing using reproducible instrumentation; and (ii) control of the delivery of sample pneumatically. MATERIALS

AND METHODS

Materials. The instrument used for pulling micropipets (Micropipette Puller M 1) was obtained from Industrial Science Associates, Inc., Ridgewood, New York. However, almost any instrument for pulling micropipets will suffice which can be adjusted for the amount of current going to the heating element and the tension of the pull on the glass capillary tubes. The uniform-bore glass capillary tubing from which micropipets are made was obtained by special order from Drummond Scientific Company, Broomall, Pennsylvania; it is type R-6 glass with an outside diameter of 0.63 mm, an inside diameter of 0.20 mm, and a length of 4 in. The original micropipet beveler (Narishige type EG-5) was manufactured by Narishige Scientific Instrument Laboratory, Tokyo, Japan. It was, subsequently, modified by the addition of: (i) a Bodine-type NSH- 12 DC motor with a Minarik No. W 14 motor speed control (Minarik Electric Company, Los Angeles, California); and (ii) a vertical post plus crossbar upon which a micromanipulator (e.g., a Narishige-type MM 333 R) is mounted (Fig. 3). These changes enable one to have a faster turntable speed and a higher degree of control over movement of the micropipet. Nevertheless, successful beveling can be obtained using the instrument as supplied by the manufacturer or comparable beveling equipment. The abrasive film (IPK 10 NO. 67777 558. alumina abrasive, 0.3 pm) used with the beveler was obtained from Arthur H. Thomas Co., Philadelphia, Pennsylvania. [LU-~‘P]GTP was purchased from New England Nuclear, Boston, Massachusetts. Experimental animals. Adult female frogs were obtained from Peter Fraser, Utica,

EQUIPMENT

FOR

MICROINJECTION

Michigan, or NASCO, Fort Atkinson, Wisconsin. The frogs were maintained under laboratory conditions for at least 2 months prior to use. Females were given 500 units of human chorionic gonadotropin (HCG)3 (E. R. Squibb and Sons, Inc.) at least 3 weeks prior to laparotomy and were used within 3 months thereafter. Only animals which had oviposited were used for the isolation of oocytes.

Treatment

and classijcation

of oocytes.

Mature oocytes (stage 6) were obtained by anesthetizing a frog in 1% TMS (ethyl-nzaminobenzoate methylsulfonate) (Sigma) for IO ~20 min followed by surgically removing one or two ovarian lobes. The oocytes were washed in Hepes-modified Barth’s solution (MBS-H) (35) and then transferred to a petri dish containing fresh MBS-H solution with IO units of K penicillin G/ml and 10 pg benzyl P streptomycin (sulfate)/ml. Stage 6 oocytes (1 1.2 mm in diameter), identified by their unpigmented equatorial band, were separated from the ovarian tissue using watchmakers forceps and a 200-~1 capillary glass micropipet. Isolated oocytes were then placed in fresh MBS-H and incubated at 19°C for 12 24 h prior to use. Pulling pipets. The pipet puller (see Materials) consists of a heating element and a pulling mechanism. Adjustable pullers like this are capable of producing glass micropipets with a wide spectrum of tip configurations. The precise shape of the pulled tip depends greatly on the geometry and temperature of the heating element and the tension applied by the pulling mechanism. Micropipets useId for oocyte injections require a gradual taper (Fig. 1B). The distance between the shaft and the tip of the pipet should ideally be 0.7 0.8 cm in length. By contrast, micropipets used for the injection of somatic cells have thin shanks and a very abruptly tapered shoulder (Fig. 1A. neuro’ Abbreviations used: HCG. human chorionic gonadotrupin: TMS, ethyl-nl-aminobenzoate methylsulfonate; Hcpcs. 4-( I-hydroxyethyl)I -piperazineethanesulfonic acid; MBS-H. Hepes-modified Barth’s solution.

OF

AMPHIBIAN

OOCYTES

301

-~ E

-h

:

3,

BOffDrn

v,e*

F~ti. I. Structures of micropipets. (A) Neurological pipet, pulled by using relatively high heat and high puller tension. (B) Oocyte microinjection pipet, made using relatively low heat and low puller tension. (C) An oocyte microinjection pipet with the tip cut at IO-pm i.d. (see Materials and Methods). (D) An enlargement of the oocyte micropipet after the beveling procedure. side view. (E) Same as D, bottom view.

logical pipet). Consequently, they are too weak for penetration through the follicular material that surrounds Xenopus oocytes. Cutting pipets. Micropipets which have been pulled as shown in Fig. 1B have a bore opening that is too small at the tip to allow for the free passage of fluids. The ends are therefore cut off to leave an inner diameter of approximately 10 pm at the tip (Fig. 1C). We perform this operation using a scalpel blade (No. 1 1) that is mounted on the mechanical stage of a microscope (Fig. 2 ). This device enables us to control accurately the movements of the micropipet relative to the scalpel blade. The apparatus consists of: (i) a simple micromanipulator (e.g., the micromanipulator that is supplied with the Narishige pipet beveler (see Materials) which is fitted with a scalpel blade and then mounted on a base plate adapted to fit the stage of a Zeiss light microscope; and (ii) a slide holder that is fitted with a rubber pad containing a slit designed to hold the micropipet firmly in place while the tip is being cut. The microscope stage and micromanipulator controls are used, respectively, to align the micropipet and the scalpel blade relative to each other while viewing them both with the microscope. Using a calibrated reticle within a 12.5X ocular of the micro-

302

STEPHENS

ET AL

-Slide

Holder -SLolpel

Assemblv Pivot

,-Pinion

Block

Assembly Assembly

,-Blade x CClnt10l

Rubber

Pod ~-

Blade

3pwlg

‘-Plnton PIi

Block

i Scalpel (Blade

Handle z Direction

Control:

FIG. 2. Micropipct tip cutting assembly. Diagram shows only the assembly attached to a Zeiss microscope stage. The objective of the microscope would be in the 2 plane directly above the micropipet tip. The micropipet is secured in the rubber pad mounted on the microscope slide (see text).

scope (total magnification, 125X), the scalpel blade is then lowered to cut off the end of the micropipet at an i.d. of 10 pm. Micropipet tips can also be cut manually using a pair of forceps, but the operation is simplified greatly and made much more reproducible using the instrument shown in Fig. 2. Beveling micropipets. Beveling a pipet is a process by which it is abraded at an acute angle to its longitudinal axis to yield a piercing tip much like that of a hypodermic needle. Beveling is the final stage in construction of micropipets and is done within a few days prior to their use. We do it using a modified Narishige-type EG-5 micropipet beveler (see Fig. 3 and Materials). The micropipet is held at a 40” angle from the horizontal by a clear plastic holder which is clamped into a micromanipulator. While visualizing the tip of the micropipet through a high-quality stereomicroscope (e.g., a Wild-Heerbrugg Model M7A stereomicroscope, total magnification 20X), the micropipet is lowered slowly onto the turntable using the micro-

manipulator. An abrasive disk is attached to the turntable (see Materials). As the turntable revolves, a bevel is gradually formed. A small amount of water added to the beveling surface at the point of contact of the micropipet tip can be used to carry away small splinters of glass that form during the beveling process. The micropipet tip is somewhat flexed as it is pressed against the beveling surface. Thus, a properly beveled pipet will have an angle of approximately 20” from the axis (Figs. 1D and E). The microinjection apparatus. The system used to empty and fill the micropipet with sample is controlled pneumatically by applying air pressure or a vacuum to the micropipet via a three-way selector valve coupled to a regulator valve containing an open end (see Fig. 4). The micropipet is placed in a microseptum fitted into a glass tubing and can be changed in seconds. Fluid is forced through the micropipet by simply placing one’s finger over the open end (finger hole) of the regulator valve. The air pressure or vacuum is then forced through the mi-

EQUIPMENT

FOR

MICROINJECTION

FIN;. 3. Micropipet beveler. The diagram indicates chassis (see Materials). The modilications include accommodation of a micropipet holder and a motor

cropipet causing the Ruid to move. There is no mineral oil present in the air or vacuum line. SamplIes are placed in siliconized conical glass ampoules made from Pasteur pipets. To fill the micropipet, the tip of the pipet is lowered into the sample solution and vacuum applied to the regulator valve. Unused sample can then be injected back into the ampoule and stored by placing Parafilm over the ampoule and inserting it into a 1.5 ml microfuge tube. Using this method, no loss of sample occurs from evaporation or from unuse’d sample left in the micropipet. For visualization of the oocytes during microinjection, a dissection stereomicroscope is used (e.g., a Wild-Heerbrugg Model M7A) (Fig. 6). The scope is mounted at approximately 30” from the vertical. The slight tilt in the microscope allows for free hand movement above the stage and the accommodation of a plexiglass shield (Fig. 7)

OF

AMPHIBIAN

OOCYTES

303

the modifications that were made to the Narishige a micromanipulator mounted at one end for the attached to the opposite end to drive the turntable.

that sets over the stage. The plexiglass shield protects the investigator from radiation exposure that might occur when injecting 32Plabeled compounds, yet does not interfere with the movements of the stage or micromanipulator. Once the microscope is focused on an oocyte, no further focusing is necessary: The stage controls are used to move each oocyte horizontally into the same field of vision while the 30” tilt of the microscope permits concurrent viewing of the micropipet tip. Thus, one’s hands are left free to move the micromanipulator vertically to puncture the oocyte and to inject the sample by placing a finger over the open end of the regulator valve. To inject, oocytes are placed with the animal pole facing upward in a 60-mm petri dish containing 12 ml MBS-H. As many as 200 oocytes can be put into one petri dish. The injections are made with oocytes sub-

304

STEPHENS

FIG. 4. Microinjection

ET AL.

stage and apparatus

merged in MBS-H. The petri dish has nylon netting adhered to the bottom to inhibit movement of the oocytes during injection. Injections are performed by: (i) manipulating the stage controls until an oocyte is directly beneath the pipet tip; (ii) puncturing the oocyte using the vertical movement of the micromanipulator to lower the micropipet (0.5 mm into the oocyte); and (iii) injecting the sample by applying air pressure (7 psi) to the pipet while viewing the meniscus through the right-angle telescope (Fig. 6). Nuclear injections are performed by injecting directly into the center of the animal pole while cytoplasmic injections are done by injecting into the animal half of the oocyte close to the equatorial band. The regulator valve is adjusted so that when not injecting there is enough positive air pressure on the micropipet to counteract

support.

See text for description.

capillary action into the tip. Since all pipets are of uniform bore no readjustment is necessary between samples. The movement of fluid is immediately responsive to placing a finger over the open end of the regulator valve. Changes in the amount of fluid in the micropipet are observed using a right-angle telescope (Figs. 5A and B) which is focused on the meniscus of fluid in the shaft of the micropipet. A lamp is placed opposite the telescope to illuminate the pipet (Fig. 6). The telescope provides a magnified image of the meniscus within the micropipet against a reticle in the ocular. Since the micropipets are made from precision glass capillary tubing, the volume of fluid delivered from a micropipet can be accurately and precisely monitored by measuring the change in the level of the meniscus against the reticle scale. Calibration of nzicropipets. [cx-~~P]GTP

-I

REF

DES

0

DESCRIPTION ACHROMAT IOOrnrn

LENS, ,I,

ELLIPTICAL 0

MAJOR

26 5mm

ROLYN MIRROR,

AXIS,

I5

DIP.

NO 200225

INCH

2 12 INCH MINOR

04

(A)

FIIG. 5. Right-angle operational telescope

telescope. (A) Dimensions and description of telescope. mounted on a platform with movable ~9 and i axes. 305

(B) Fully

assembled

and

306

STEPHENS

FIG.

6. Completely

apparatus

are

assembled

shown

apparatus. (A) Stage of the microinjections. angle telescope used on the selector

here:

microinjection

the

microscope

ET

apparatus. has

been

moved

AL.

The

four

to the

major side

parts

of the

to allow

microinjection

a fuller

view

of the

and apparatus support (see Fig. 4). (B) The stereomicroscope used for visualization (C) The lamp (see Materials) used to illuminate the micropipet. (D) The rightto magnify and visualize the meniscus of fluid within the micropipet. Contained

stage and apparatus support are the following: (E) the micromanipulator. value; (G) the regulator valve; and (H) the ledge used to support the

of known specific activity was injected into oocytes to calibrate accurately the amount of fluid being delivered. By allowing the meniscus in the micropipet to travel a predetermined number of graduations on the

(F)

the

three-way

lamp.

telescope reticle scale, a direct correlation between distance traveled on the reticle scale and volume of fluid injected can be determined. Oocytes injected with [(u-“P]GTP were dissolved in NCS tissue solubilizer (Amersham) and then counted in OCS toluene-based scintillation cocktail (Research Products International). RESULTS

FIG. 7. Plexiglass shield. The shield is shown mounted over the stage assembly (see Fig. 4). The shield as made does not interfere with movement of either the stage or the and

micromanipulator. is 7 in. high.

The

shield

is 4%

in. at the

base

The microinjection apparatus was calibrated (see Materials and Methods) such that each reticle division observed in the right-angle telescope corresponded to one nanoliter of fluid being delivered from the micropipet. The results obtained from injecting 5, 10, 25, or 50 nl are shown in Table 1. The actual mean volume introduced into 10 oocytes was within 9% of the expected value for 5-nl injections and within 558% of the expected value for injections from 10 to 50 nl. As expected, precision of volume delivered increased with increasing volume. The standard deviation was 4---6% of the mean for volumes in the range lo-50 nl and

EQUIPMENT

FOR

TABLE ACTI

(\I

Vot.uvts

Ttl1:

I

INJFCXD

INTO

CALIHRATFD

(nl)

SD Percentage error SD as percentage of the mean ” The

reticle

volume

delivered --~

5

IO

5.42 0.47

10.54 0.61

23.00 1.19

8.4

5.4

8.0

5.9

8.7

5.8

5.2

4.0

of the

that one division actual volumes

by counting Materials from

USIM; A

25

50

volume

dispensed

such The

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RFTICIM” Expected

Mean

OWYTKS

F TEI.~:SCOPE

RIGHT-ANGI

MICROINJECTION

and

right-angle

the amount Methods).

IO InJected

telescope

is equal to one of fluid dispensed

52.91 2.1

is calibrated

nanoliter of fluid. were determined

of “P present per Each determination

oocyte (see was made

oocytes.

approximately 9% of the mean for volumes in the 54 ramge. DISCUSSION The type of apparatus used for microinjection of amphibian oocytes should be simple enough to enable a beginning investigator to obtain reproducible injections with a minimum of practice. The instrumentation described here for the construction and use of micropipets is relatively inexpensive and can be modified to fit a particular laboratory’s needs. The basic components needed for microinjection are a right-angle telescope or other device for viewing the meniscus of fluid in the micropipet, a stereomicroscope for visualizal.ion of the injections, air and vacuum sources, a three-way valve, a onedimensional micromanipulator, a simple regulatory valve, and a stage that can hold and position a petri dish. The construction of good micropipets is also crucial to successful microinjections. The basic equipment needed for the construction of lnicropipets are a puller and a beveler. Pipets can be cut manually but it

OF

AMPHIBIAN

OOCYTES

307

is simpler and more reproducible to use an instrument such as the one described in Fig. 2. There are several points to be stressed in constructing micropipets. Micropipets cut with an inside bore diameter of approximately 10 pm are small enough to allow easy penetration of the oocyte membrane and surrounding follicular material. Larger tips may cause excessive damage to the oocyte. A proper bevel at the tip enables the micropipet to penetrate the oocyte easily, resulting in a minimum of damage. Fluid will flow quite readily through a IO-pm i.d. tip, which will normally allow a flow rate of approximately 10 nl/s. A micropipet with a properly beveled tip can be refilled and used for as many as 200-300 injections without acquiring blockage. With a minimum of practice, one can easily inject approximately 200 oocytes per hour. Microinjections performed with properly constructed pipets cause minimal damage to oocytes. The number of oocytes which survive after a single injection is greater than 95% and greater than 90% after a second injection. The hole that is left in the oocyte membrane following injection with a properly constructed pipet seals well without allowing much leakage of material from the oocyte. Oocytes microinjected with [CY“P]GTP retain greater than 95% of the label within the oocyte, with less than 3% being found in the follicular material (data not shown). Gurdon (19) has reported that when injecting volumes in the range 10-100 nl there is a ? 10% error in the calibration of micropipets. However, operator judgement will vary in calibrating the micropipets and varies with experience. Therefore, the final error in volume may be greater than 10%. Hitchcock and Friedman (36) describe a technique using an automated device for controlling the volume of injection. They claim that volumes in the range lo-50 nl are within 10% of the expected value and 20% for I-n1 injections. They also reported stan-

308

STEPHENS

dard deviations of 10P 15% of the mean for volumes in the range 30-50 nl and 30-40s of the mean for volumes in the range l-2 nl. In the system reported here each pipet has the same inner bore diameter; once a micropipet is calibrated using the reticle scale of the right-angle telescope, no further calibrations are needed to determine the accuracy or precision of the volume injected with subsequent micropipets. Delivery of different volumes is easily increased by simply allowing more fluid to pass while viewing the meniscus through the right-angle telescope. As shown in Table 1, the volume of injected material is within 9% of the expected value for all volumes tested, and the standard deviation is less than 6% of the mean for volumes from 10 to 50 nl. Many types of experiments require microinjection of samples into the nucleus of the oocyte (23). However, the nucleus of a Xenopus oocyte is only approximately 50 nl in volume. Consequently, accurate injection of volumes of 10 nl and less is frequently important, because microinjection of greater than 20 nl of fluid may disrupt the nucleus. Success in performing nuclear injections depends on the ability to penetrate the center of the animal pole. When performing nuclear injections as described above, we routinely inject samples successfully into the nucleus of Xenopus oocytes approximately 70-80% of the time (data not shown). The greatest advantage of the microinjection equipment described here is that inexperienced personnel can learn to perform reproducible injections within a short period of time. The injection procedure requires only the ability to puncture an oocyte using a stationary, one-dimensional micromanipulator and then to deliver the sample by placing a finger over a hole in a regulatory valve (Fig. 4) while watching the movement of the fluid through a right-angle telescope. ACKNOWLEDGMENTS We are indebted to Gunther Albrecht-Buehler for designing the right-angle telescope described in this pa-

ET AL. per and to Robert Benbow for discussions. This work was supported by U. S. Public Health Service Research Grants CA 07175 and CA 22443 from the National Cancer Institute. T.J.M. was supported by U. S. Public Health Service Training Grant CA 09075 and a feilowship from the American Cancer Society, PF- 1678.

REFERENCES I. Lane, C. D., Marbaix,G.,andGurdon. J. EL (1971) J. Mol. Biol. 61, 73-9 I. 2. Gurdon, J. B., Lane, C. D., Woodland, H. R.. and Marbaix. G. (1971) Nature (London) 7.33, l77182. 3. Reynolds. F. H., Premkumar, E., and Pitha, P. M. (1975) Proc. Nat. Acad. Sri. USA 72, 48814885. 4. Berger. S. L., Hitchcock, M. J. M.. Zoon. K. C., Birkenmeier, C. S., Friedman, R. M., and Chang, E. H. (1980) J. Biol. Chem. 255, 2955-2961. 5. Asselbergs, F. A. M., Van Venrooij. W. J., and Bloemendal. H. (1978) Eur. J. Biochem. 87. 5 17-524. 6. Beato, M., and Rungger, D. (1975) FEBS Lert. 59, 305309. 7. Vassart, G., Brocas, H., Lecocq, R., and Dumont, J. E. ( 1975) Eur. J. Biochem. 55, 15-22. 8. Gedamu, L., Dixon, G. H., and Gurdon, J. B. (1978) Exp. Cell Res. 117, 325-334. 9. Rapoport, T. A.. Thiele, B. J., Prehn, S.. Marbaix, G., Cleuter. Y., Hubert. E., and Huez, G. (1978) Eur. J. Biochem. 87, 229-233. IO. Kindas-Mugge, I., Lane, C. D., and Kriel. G. ( 1974) J. Mol. Biol. 87, 45 l-462. II. Larkins, B. A., Pedersen. K.. Handa, A. K.. Hurkman, W. J.. and Smith, L. D. (I 979) Proc. Nat. Acad. Sci. USA 76, 6448-6452. 12. Katz, R. A., Maniatis, G. M., and Guntaker, R. V. (1979) Biochem. Biophys. Res. Commun. 86, 447-453. 13. Dicou, E.. Brachet, P., Huez, G., and Marbaix, G. (1979) FEBS Lett. 104, 275-278. 14. Laskey, R. A., Gurdon. J. B.. and Crawford. L. V. (I 972) Proc. Naf. Acad. Sri. USA 69, 366% 3669. 15. DeRobertis, E. M., and Mertz, J. E. (1977) Cell 12. 175-182. 16. Rungger, Acad.

D.. and Turler, H. (1978) Sci. USA 75, 6013-6077.

Proc.

NUZ.

17. Gurdon, J. B., Lingrel. J. B., and Marbaix. G. (1973) J. Mol. Biol. 80, 539-551. 18. Mach. B.. Faust. C., and Vassalli, P. (1973) Proc. Not. Acad. Sri. USA 70, 451-455. 19. Gurdon, J. B. (I 974) The Control of Gene Expression in Animal Development, Oxford Univ. Press, London.

EQUIPMENT

FOR

MICROINJECTION

20. Zehavi-Willner, T., and Lane, C. (1977) Cell 11, 6833693. 21. Lane, C.. Shannon, S., and Craig, R. (1979) Eur. J. Biochem. 101, 4855495. 22. Coleman, A.. and Morser. J. (1979) Cell 17, 517~ 526. 23. Mertz. J. E.. and Gurdon. J. B. (1977) Proc. Nat. .&-ad. Sri. USA 74, 1502-l 506. 24. Brown, D. D., and Gurdon. J. B. (1977) Proc. Nat. Acad. Sci. USA 74, 2064-2068. 25. Kressmann. A., Clarkson. S. G.. Pirrotta, V.. and Birnstiel. M. L. (1978) Proc. Nar. Acad. Sci. us.4 75, I 17661 180. 26. Wyllie. A. H , Laskey, R. A., Finch, J.. and Gurdon, J. B. (1978) Develop. Biol. 64, 178-188. 27. Gurdon. J. B,., and Brown, D. D. (1978) Develop. Biol. 67. 246-356. 28. Trendelenburg. M. F.. and Gurdon. J. G. (1978) IVUIUW (London/ 276, 292-294.

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OOCYTES

29. Laskey. R. A., Honda, B. M., Mills, A. D., Morris, N. R., Wyllie. A. H., Mertz. J. E., DeRobertis, E. M., and Gurdon, J. B. (1977) Cold Spring Harbor S-vmp. Quant. Biol. 42, I7 I~-1 78. 30. DeRobcrtis. E. M.. and Olson. M. V. (1979) Nafurc (London] 278, 137-143. 31. Probst. E.. Kressmann. A., and Birnstiel. M. L. (1979) J. Mol. Biol. 135, 7099732. 32. Melton, D. A.. and Cortese, R. (1979) Cell 18, 1165-I 172. 33. Elsdale, T. R., Gurdon. J. B., and Fischberg, M. (I 960) J. Embryo/. Exp. Morphol. 8, 437-444. 34. Gurdon, J. B. (1977) in Methods in Cell Biology (Prescott, D.. ed.). Vol. 16, pp. 1255140, Academic Press. New York. 35. Gurdon, J. B. (1976) J. Embryo/. 36. 523-540. 36. Hitchcock, M. J., and Friedman, Anal. Biochenz. 109, 338-344.

Exp.

Morphol.

R. M.

(1980)