Improved system for capillary microinjection into living cells

Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/07003 l-07$02.00/0

Experimental

IMPROVED

SYSTEM

Cell Research 140 (1982) 31-37

FOR CAPILLARY

INTO

LIVING

MICROINJECTION

CELLS

W. ANSORGE European Molecular

Biology Laboratory,

D-6900 Heidelberg,

Germany

SUMMARY A technique for microiqiection into cells is described, with several simplifications as to preparation and handling of micropipettes, sample filling and injecting system. The simplified procedure enables us to pull and fill the capillaries in an easy, reproducible and fast way, requiring only about 1 min. The injecting system uses three positive pressure levels (high, injecting, holding) controlled from one button. It provides for quick and reproducible changes among the three pressures. Furthermore, it limits the chances for clogging the microcapillary by elimination of the temporary negative pressure (suction at its tip), which would normally occur when the pressure is reduced from the injecting to a zero (atmospheric) pressure value. The estimated sample ejection rate which is achieved with this system could correspond to 10-1’-10-‘3 ml/set, according to [6]. Compared with other known techniques using pressurized gas in bottles, this ejected volume is three orders of magnitude smaller and makes the system of interest for work with cells smaller than 50 pm. Whereas the sample volume injected into cells is reproducible, further improvement in the control of the amount actually injected is desirable. The manipulation of the sample ejection system is very easy and no time is needed to learn it. The user can inject the sample volume either in the cytoplasm or the nucleus, as desired, by manipulating a single button. Operational aspects and problems with capillary clogging are discussed. The reliability of the whole system and technique is shown in the results obtained on microinjection of living cells. Cells could be injected at an initial speed of about 600-800 cells per hour, the actual number of cells injected in 1 h being about 400. After injection of SV40DNA, 70-80% of the injected cells have synthesized the T-antigen. The above improvements make the injection technique easily accessible to molecular biologists who need to screen for expression of the recombinants made in vitro, or to study the distribution of fluorescently labelled structural proteins. The pressure, the valves and the penetration of the cell (piezo driver) may be regulated electronically. The system would be of interest for any automated injection process. When the common pressure regulator (from 150 to 1 bar) is available, the cost of the additional equipment shown in fig. 2b is as low as 500 DM.

The microinjection technique [l-7] has proved to be a useful addition to the tools available for tests on biological material at the cellular level. Impressive results were reported in the references cited. The technique (as described in [6]) has been worked out in detail and works well in the hands of well trained and experienced persons. Whereas locating the cell and manipulating the micropipette under a microscope remains a matter of practice, we tried to 3-821808

simplify the other factors affecting the microinjection process: capillary pulling, its filling with the sample and ejection of the sample. The micropipettes are prepared directly on the puller without manual prepulling of the glass capillaries. The sample is filled through the rear open end of the micropipette by means of X-ray glass cylinders available commercially. Change of a capillary through the two steps is accomplished in about 1 min. Exp Cell RPS 140 (1982)

32

W. Ansorge

Injection in nuclei or cytoplasm is performed using an ejection system with three pressure levels, manipulating a single button. Practically no time is needed to learn how to use it. MATERIALS

AND METHODS

A phase-contrast inverted microscope (Leitz or Zeiss) was used with x250-640 magnifications. Capillaries with a thin glass filament were from Clark Electromedical Instruments (Pangboume, Berks, UK, 10 cm long, GC-120-F-10 0 1.2 mm, cat. no. GC-IMF-10). Washing can be done as in [5], or the capillaries are washed in ethanol for 1 h or longer, drained and dried at 130°C for 30 min, prior to pulling. The capillaries (or their tips after pulling) can be made hydrophobic by dipping them in a mixture of ether and silicon solution from Serva (ratio 3 : 1) and baking for 1 h at 130°C.

Micropipette

preparation

Without manual pre-pulling, the glass capillaries are directly inserted and clamped in the puller (E. Leitz, Wetzlar. FRG). We are usine the minimum force setting available on the puller,?.e. the lowest tension in the spring. The temperature of the platinum tilament is determined by -the electrical current passing through from the transformer. The current is maintained at about 5.9 A during pulling. The capillary must not touch the filament. Micropipettes are prepared reproducibly in this way, as seen under the microscope, with the tip diameter around 1 pm. It is possible to draw pipettes with diameters below 1 pm.

Filling

and manipulation

of micropipettes

After pulling, the capillary is placed in a support (tin. 3) which protects the tin from breakage durine filhng of the sample. The sample is delivered through its rear open end down near to the tip by means of an X-ray glass cylinder (0 0.1 or 0.2 mm; A. Mueller Glas- und Vakuumtechnik. Berlin). These elass cvlinders can be cleaned in the same way asdeschbed above for the micropipettes. If the sample is not deposited down at the tip, but higher in the pipette, it will still be drawn to the tip due to the capillarity effect provided by the glass filament. However, as it descends to the tip, the walls ‘of the micropipette are washed by the sample, and the risk of collecting any remaining dust particles and blocking the capillary increases. Usually about 0.1-l ~1 of sample volume (well centrifuged, about 10 min at 1OOOO e or h&her). at a DNA concentration of 0.02-l mg/mlr is tillid into the capillaries. By means of another X-ray glass cylinder, diameter 0.5 mm, A. Mueller Glas- u. Vakuumtechnik, a column of one or more centimeters height of heavy paraffin oil, Merck, is layered on top of the sample. The bottles containing the compressed gas Exp Cell Rcs 140 f 1982)

(nitrogen, air) are opened to pressurize the system (see below) and the tilled micropipette is removed from its support and inserted into the capillary holder of the micromanipulator, E. Leitz, Wetzlar. When mounted in the holder, the capillary tip is submerged in the cell medium to avoid drying of the sample in the air, which may lead to a blocked capillary. When a very minute (less than 0.1 ~1) volume of a sample is available. it may be tilled into the micropipette from its tip, as described in [5].

Sample ejection system (Fig. 2~). When the filled capillary is submerged in the cell medium, the sample is pushed through the narrow tip of the pipette by applying a high pressure P3 (button A pressed all the way down to position 3, giving about 2 bars, or as adjusted). High sample flow can be observed under the microscope. When the pressure button A is released completely (position 1) a small positive holding pressure Pl (about 0.03 bar or as adjusted, see below) is set in the capillary. The holding pressure prevents the fluid from re-entering the tip of the capillary. As the micropipette approaches the cell to be injected, the pressure button A is pressed half-way down till resistance is first encountered (position 2). In this position the injecting pressure P2 (about 0.12 bars or as adjusted) is set in the capillary. The pipette is introduced into the cytoplasm or the nucleus, as desired, and the sample is injected at the flow rate corresponding to pressure P2. The amount injected can be controlled by the length of time that the tip remains inside the cell and the injection may be visually assessed. Before releasing the pressure button A back to the holding pressure (position 1) to stop the injection, the micropipette is taken out of the cell, in order to limit the possibility of clogging the tip by reentering fluid while the injecting pressure is lowered. This is not necessary if the holding pressure Pl is properly adjusted (no re-entering fluid) and the injection may be stopped while the tip is still in the cell. Whenever there is doubt as to the running conditions or when the capillary seems to be clogged,. the tip is taken out of the cell and the flow condtttons may be restored by a short application of the high pressure P3. The typical values for pressures Pl, P2, P3 given in this section were for sample concentrations of 0.1 mg DNA per 1 ml fluid. The system is designed to quickly produce the pressure value desired when changing between three pressure levels. The gas is discharged through the depressurizing outlet of-button A, when this isreleased cornpletely. In this position “1” the pressure in the capillary is lowered from P2 to a reduced level PI bv aas leakage through button A (or fig. 26, button A2): fhe leakage flow (and the holding pressure Pl) can be regulated using a valve with continuous flow regulation (valve CV in tig. 2) as shown. The pressures P2 and Pl can be read on manometer M (fig.-26). The unidirectional blocking valve BV protects the low pressure regulator while the high pressure is on the line. When desired, the line pressure can be reduced to a fourth pressure (atmospheric) while pressing but-

Improved system for capillary microinjection

33

Fig. I. View of the microinjection set-up. A, pressure button A; B, pressure button B; CV, continuous flow regulation valve; D, bottle with CO, (for pH regulation); E, microscope; F, micropipette holder; G, mi-

cromanipulator; H, injecting system (partially seen); I, balance table (vibrations damped by a pad of hard rubber on the floor).

ton B. An additional pressurizing or vacuum unit (pressure delivering pump, suction pump, syringe . . .) can be attached to the system at the connection P, closing or opening valves V2 or V3. The low pressures P2 and PI may be produced also by a small pump, e.g. an aquarium pump (WISA, Wuppertal, FRG), with both pressure and suction terminal available. If there is fear of applying inadvertently high pressure P3 instead of injecting pressure P2 and bursting the cell, it is possible to control the injecting and holding pressure from one button (fig. 2b, button A2) and, separately, the high pressure (button A). Button A2 is connected with polarity opposite to that of button A, i.e. it closes the line when pressed. In fig. 2b a system using only one gas bottle is also shown. The injecting system (fig. 2) uses bottles with commessed gas (air or nitrogen) for adiustment of the high pressure-P3 and injecting pressure P2 (Messer G&sheim. FRG; part nos MD-622-3 and MD-652-3 are the high and low-pressure regulators, respectively). Other regulators were also used. The orecision low nressure regulator LP in rig. 26 is a Fairchild Model 10 (in Germany available from Pressluft Goetz GmbH, Mannheim).

All parts, line and connections of the pneumatic circuit were built from standard parts obtained from Festo, Esslingen, FRG. Buttons A and A2 are product no. SV-3-MS-P22, the blocking valve BV no. H-l&, valve CV no. GRO-l/4 or any precision valve. Valves Vl, V2, V3 may also be any ON/OFF ventils. When properly mounted, the system can withstand pressures up to about 7 bars. Button A2 may be a foot switch, no. F-3-MS, freeing both hands for micromanipulation.

RESULTS To test the viability of the designed system, DNA of Simian virus 40 (SV40) was injected into the nucleus of monkey (CVl) cells. As known [e.g. 61, the early phase of infection with SV40 is marked by the synthesis of a tumor antigen (T-antigen) which is accumulated in the cell nucleus. Cells Exp Cell RPS 140 (1982)

34

W. Ansorge

HIGH PRESSURE P) REGULATOR

LdA BE:

Fig. 2. (a) Schemeof the injecting system. (For ex- (b) injecting systemusing only one gasbottle. Button planations, see text.) Button A: Position 1, holding A2 controls injecting pressure,button A controls high pressure; 2, injecting pressure; 3, high pressure. pressureP3(further explanationsin text).

were fixed 24 h after they were injected, exposed to anti-T antibody and then to a second fluorescently labelled antibody. Fig. 4 shows a photograph of the cells after the treatment. Fluorescence is visible only in the nuclei, indicating the presence of Tantigen. One can conclude that the cells were not affected by the injection procedure. The T-antigen was synthesized efficiently, as it is observed when DNA is introduced by microinjection [6]. The presence of T-antigen was observed in 7&80% of the injected cells. DISCUSSION The results have proved that the cells were surviving perfectly well the injection with the system described above. The simplified preparation of micropipettes, drawn directly on a puller without prior manual pre-pulling, gives excellent reproducibility in their dimensions, as viewed under the microscope. The filling of the pipettes from their rear open end, during which the sample is deposited by means of X-ray glass cylinders at the tip in a few seconds, has also proven to be a valuable simplification. There is no waste of sample due to its evaporation, as can be the case in

the standard technique when the capillary is filled very slowly from the tip in the atmosphere. Both of the above simplifications in the procedure are also much less timeconsuming, compared with the standard [l63 procedures. It takes about 1 min to prepare, till and exchange a capillary, when needed. A difficulty, occurring often mainly in the learning phase, is the clogging or breakage of the microcapillary. In our experience, besides false micromanipulation (sweeping the tip through the cells), the following were the main causes of the clogging: (1) Suction of the fluid through the tip (lowering the pressure from the high value P3 while the tip is still in the cell, when the

rubber

l, ,[

-t;’

bond

swvrtlng bhk

3. Support for the micropipette during sample tilling.

Fig.

Improved

system for capillary

microinjection

35

Fig. 4. Injected cells viewed by fluorescent microscopy.

holding pressure Pl is not correctly ad- lead to capillary clogging, is caused by a temporary negative pressure. The liquid justed). (2) Particles in the sample. This may be from the surroundings is drawn inside the capillary through the fine tip, which can get the case when the sample is not sufficiently centrifuged or when the capillary con- blocked by particles from the cell culture tains dust. When filling the sample, it is medium or by cell organelles. Negative advisable to deposit it at the tip as men- pressure can occur in the capillary when the tioned above, so that it does not wash off pressure level is changed to zero (atmospheric). This is one of the reasons why the the capillary walls as it moves. (3) Drying of the sample at the tip, if the syringe ejection technique [6] seems to require a lot of practice, because it is not so filled capillary is not submerged within easy (with the very small sample volumes) about 2 min in the fluid. The breakage of the tip is caused mainly to control the piston position, when lowerby faulty micromanipulation or focussing, ing the pressure to stop the injection, and when the micropipette hits the surface of not to create a negative pressure. The techthe dish, and sometimes after pulling while nique of continuous slow ejection is often the solution of this problem, if it can be afhandling the capillary. forded. In the other methods using comThe suction in point 1 above, which may Exp Cell RPS 140 11982)

36

W. Ansorge

pressed air, as they are mentioned in the excellent review in [8], the injected sample volumes being thousand times larger, the aspect of temporary suction when switched to zero pressure is not considered. In the injecting system described above, the problem is solved by the provision of the holding pressure Pl, as shown. Ideally, the low holding pressure setting can be chosen so as to just hold the sample fluid in the micropipette tip when the pressure is reduced from the injecting to the holding level, so that the injection may be stopped while the tip is still in the cell. However, a precise detection of zero sample flow is not an easy task, even at very high magnifications. Suitable settings for injecting pressure P2 and holding pressure Pl are found in practice by testing the given sample with its viscosity on cells chosen for the experiment. If slow continuous sample ejection mode is desired, the button is held pressed in position 2 with little effort, or it can be locked in that position, or the valve CV is closed. The amount of injected sample in the given cell compartment is determined by the length of the time during which the capillary remains in the cell and by the injecting pressure P2. As the results have shown, the sample volume injected was reproducible from cell to cell. The amount injected per second (estimated according to [6]) was about 10-l) ml. Smaller (ten to hundred times) injection rates were achieved with the system. While the reproducibility is good, further improvement in the control of the actual amount injected is desirable. With some training in the micromanipulation it was possible to inject on the simplified system with an initial rate of 600400 cells per hour. The actual number of cells injected in 1 h was about 400. Exp CdRcs

140 f/982)

The advantage of the system is the prevention of fluid re-entry in the capillary tip (avoiding both capillary clogging and dilution of the sample in the pipette), availability of different adjustable pressure levels (which may present a problem with injections using syringes) and their simple command, providing a quick and reproducible pressure application. With the obtained low sample volumes, it is possible to work with very small cells. The number of cells injected with a single pipette is virtually limited only by the sample volume filled in the pipette. Practically no time is needed to learn how to use the ejection system. The pressure, the valves and the penetration of the cell (piezo driver) may be regulated electronically. The system would be of interest for any automated injection process. When the common pressure regulator (from 150 to 1 bar) is available, the cost of the additional equipment shown in fig. 2b is as low as 500 DM. The injection technique described above is suitable for studies on the distribution of fluorescently labelled structural proteins and for screening of cloned recombinants. It is easily mastered even by an inexperienced operator and is more efficient and reproducible than the commonly used calcium phosphate coprecipitation technique [91. The author thanks Dr A. Jones and professor K. Simons for their support in the course of the work, and drs P. Oudet and M. P. Gaub at the Laboratoire de Genetique Moldculaire des Eukaryotes in Strasbourg for assistance in the evaluation of performance and results obtained with the new system.

REFERENCES 1. de Fonbrune, P, Technique de micromanipulation. Monographies de I’Institut Pasteur, Masson et Cie, Paris (1949). 2. Diacumakos, E G, Methods in cell biology 7 (1973) 287.

Improved system for capillary microinjection

37

3. Anderson, W F, Killos, L, Sanders-Haigh, L, 7. Capecchi, M R, Cell 22 (1980) 479. Kretschmer, P J & Diacumakos, E G, Proc natl 8. Taylor, D L & Wang, Y L, Nature 284 (1980) 405. acad sci US 77 (1980) 5399. - 9. Chu, G & Sharp, PA, Gene 13 (1981) 197. 4. Graessmann, M & Graessmann, A, Proc natl acad sci US 73 (1976) 366. 5. Stacey, D W & Allfrey, V G, Cell 9 (1976) 725. Received July 27, 1981 6. Graessmann, A, Graessmann, M & Mueller, C, Revised version received January 29, 1982 Methods in enzymoI65 (1980) 816. Accepted February 1, 1982

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in Sweden

Exp Cd/ Rcs 140 (1982)