Instruction Manual

™

CELL PORATOR® ElectroporationSystem I, 230 V

CAT. NOS. 71600-050 and 11609-039

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Table of Contents

1. Notices to Customer .............................................................................. 11.1 Important Information................................................................................ 11.2 Warnings................................................................................................... 1

2. Overview ..................................................................................................... 22.1 Description................................................................................................ 22.2 Components ............................................................................................. 32.3 Overview of Electroporation...................................................................... 6

2.3.1 Background Information .................................................................. 62.3.2 Electrical Principles ......................................................................... 72.3.3 Electroporation Parameters ............................................................ 8

1. Optimizing Field Strength and Pulse Length .............................. 82. Setting and Measuring Pulse Voltage ........................................ 83. Determining Pulse Length .......................................................... 84. Cell Survival Rate and Transformation Efficiency .................... 11

3. Operating Instructions ....................................................................... 123.1 Procedural Overview .............................................................................. 123.2 Preparing Cells for Electroporation......................................................... 12

3.2.1 Mammalian Cells........................................................................... 123.2.2 Plant Cells ..................................................................................... 13

3.3 Setting Up the Work Area ....................................................................... 133.4 Electroporation........................................................................................ 13

3.4.1 Equipment Set-Up......................................................................... 133.4.2 Electroporation .............................................................................. 143.4.3 Post-Electroporation...................................................................... 16

4. Troubleshooting Guide ....................................................................... 174.1 General Troubleshooting Guidelines ...................................................... 174.2 Pulse Control Checkout .......................................................................... 18

4.2.1 Basic Controls ............................................................................... 184.2.2 Pulse Discharge Functions ........................................................... 184.2.3 Oscilloscopic Analysis................................................................... 18

5. References and Select Bibliography ............................................ 205.1 References ............................................................................................. 205.2 Select Bibliography ................................................................................. 20

6. Related Products ................................................................................... 23

7. Additional Information ........................................................................ 247.1 Care and Handling.................................................................................. 247.2 Maintenance ........................................................................................... 247.3 Specifications.......................................................................................... 247.4 Warranty ................................................................................................. 257.5 Declaration of Conformity and CE Mark ................................................. 25

Appendix: Experimental Data ................................................................ 26

Figures1. CELL-PORATOR Electroporation System I .......................................................... 22. Pulse Control Panels ....................................................................................... 33. Chamber Safe, Chamber Rack, Pulse Cable, and

Disposable Electroporation Chambers ............................................................ 44. Top View of Chamber Safe.............................................................................. 55. Basic Design of an Electroporation System..................................................... 66. Capacitor Discharge Curve.............................................................................. 77. Schematic Diagram of CELL-PORATOR System Pulse Circuit............................ 88. Operating Arrangement for the CELL-PORATOR System.................................. 13

Tables1. Pulse Length for HEPES-buffered Mannitol (HBM) Solution at 0°C ................ 92. Pulse Length for HEPES-buffered Saline (HBS) Solution at 0°C .................... 93. Pulse Length for HEPES-buffered Mannitol (HBM) Solution at 22°C ............ 104. Pulse Length for HEPES-buffered Saline (HBS) Solution at 22°C ................ 10

CELL-PORATOR®, HORIZON®, FOCUS®, NACS™; TECH-LINESM, and the Life Technologies logo are marks of LifeTechnologies, Inc.

Warning:

1. Read and carefully follow manual instructions.

2. Do not alter equipment. Failure to adhere to these directions could resultin personal and/or laboratory hazards, as well as invalidate equipmentwarranty.

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Notices to Customer

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1.1 Important Information

This product is authorized for laboratory research use only. The product has notbeen qualified or found safe and effective for any human or animal diagnostic ortherapeutic application. Uses for other than the labeled intended use may be aviolation of applicable law.

1.2 Warnings

1. DANGER! Although designed to prevent accidental exposure to high voltages,this apparatus should always be operated with extreme caution. Do not attemptto defeat safety interlock features designed for user protection. Carelesshandling could result in a lethal electric shock. This system should be operatedby trained personnel only. This manual should be readily accessible to allusers.

2. Never operate damaged or leaking equipment.

3. DANGER! Do not leave the system charged when not in use. Discharge thepulse control as instructed in Chapter 3, and turn the system off beforeremoving the pulse cable.

4. Avoid spilling liquid on or into the apparatus. Do not submerge the apparatus inany liquid. Do not operate the apparatus if it is accidentally wetted.

5. Do not open the instrument case of any CELL-PORATOR System component.These apparatus contain no user-serviceable parts.

6. Do not change the CAPACITANCE selector switch while the capacitors arecharged >10 V. Repeated switching with charged capacitors will damage theswitch.

7. Do not use this system under pulse conditions generating a current of >40 Amperes (A) with a pulse length >2 ms. Damage to the chamber safe mayresult.

8. Always connect the CELL-PORATOR System to a 3-wire, grounded AC poweroutlet. Do not alter the grounded AC power cord provided with the unit.

9. For maximum safety, always operate this system in an isolated, low-traffic areathat is not accessible to unauthorized personnel.

10. Before operating this system, make sure that all electrical connections aresecure and that power cords show no signs of damage.

11. Certain reagents indicated for use in this manual are of a hazardous nature.The researcher is cautioned to read the Material Safety Data Sheets (MSDS)which are provided by the supplier for these reagents. Care must be exercisedin the proper use of the equipment used in these procedures, always followingthe manufacturers' safety recommendations.

12. If this equipment is used in a manner not specified in this manual, theprotection provided by the equipment may be impaired.

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Overview

2.1 DescriptionThe CELL-PORATOR® Electroporation System I is an electroporation device designedto provide a reproducible, safe, and convenient way to introduce DNA intomammalian or plant cells. This manual provides instructions for proper setup andoperation of the CELL-PORATOR Electroporation System and its components, alongwith background information on electroporation and parameters for designingexperiments with the CELL-PORATOR System. Details of experiments and data for theelectroporation of mammalian and plant cells are provided in the appendix.

For greatest assurance of success, read this manual in its entirety prior toperforming experiments with the CELL-PORATOR System. Users new to the field ofelectroporation should also familiarize themselves with the literature cited in theSelect Bibliography in Chapter 5.

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Figure 1. C ELL-PORATOR Electroporation System I.

Chamber Safe

Disposable Electroporation Chamber

Chamber Rack

Fi 1 C P El i S I

Pulse Control with internal power supply

TRIGGERDOWN

UPARM

HARGE

POWER

Cell—Porator

Chamber Rack

™

Cell—Porator®

DC VOLTS

PULSE CONTROL+POWER SUPPLY

1

2

4

3

CAPACITANCE(µF)

800 118000

80

330

0

0

CHARGE RATE

MED FASTLOW

HIGH ΩLOW Ω

2

3

Overview

Figure 2. Pulse Control Panels.

2.2 ComponentsThe CELL-PORATOR Electroporation System I is designed to function as a complete,integral system, providing the highest possible degree of safety and conveniencefor electroporation experiments. Each system component is engineered fordurability and ease of use. Refer to figure 1 to identify the following features andcomponents:

• Pulse Control (with internal power supply)• Chamber Safe• Chamber Rack• Disposable Electroporation Chambers• Pulse Cable*• Instruction Manual*

Many of these components are also available separately. For ordering information,refer to Chapter 6.

* not shown

CHARGE/ARM switch Voltage controls Pulse discharge TRIGGER button

Pulse indicator

Capacitor CHARGE RATEswitch

HIGH /LOW switch

LCD voltmeter

CAPACITANCEselector switch

POWER indicatorlight

ARM indicator

Pulse output connector Oscilloscope outlet

Main POWER switch

AC power input

light

lightHandle/stand

Handle lock screwLIFE NOLOGIESTECH

Pulse Control+Power SupplyELLC -PORATOR

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Figure 3. Chamber Safe, Chamber Rack, Pulse Cable, and Disposable Electroporation Chambers.

Chamber Safe

Draw latch

Disposable Electroporation Chamber

Cell—Porator

Chamber Rack

1

2

4

3

Contact rivets

Pulse Cable

Chamber slotChamber Rack

Lift tab

Aluminumelectrodes

Safety interlock lid

Spring-loaded contact

Temperature control compartment

The pulse control (figure 2) delivers electrical pulses to cells by capacitor discharge.Capacitance is adjustable with eight sett ings ranging from 10 to 1,980 µF. This unit features an internal power supply capable of generatingvoltages up to 400 VDC. When used with disposable electroporation chambers, theunit delivers pulses with a field strength of up to 1,000 V/cm and a pulse length thatis variable from 0.10 to 200 ms with commonly used buffers. The unit has a SCOPEoutlet located on the rear panel for monitoring pulse characteristics with anoscilloscope. The symbols used on the mains power switch are defined as follows:

ON is indicated by the symbol “1” (mains power connected to unit).OFF is indicated by the symbol “0” (mains power disconnected from unit).The stand (figure 1) can also be used as a handle for the unit.

The pulse control is equipped with a capacitor CHARGE RATE switch for variablespeeds to charge or discharge the capacitors, as well as a HIGHV/LOW V switchfor use with sample buffers of different conductivity. The LOW V position is used fora low-resistance medium (for example, phosphate-buffered saline). The HIGH Vposition is used in a system that employs a high-resistance medium (for example,mannitol). In the HIGH V position, a 10 V internal resistor is connected in parallelwith the disposable electroporation chamber, providing a means to generateacceptable pulse lengths with these buffers. The pulse control has a single pulsedischarge TRIGGER button for safe delivery of the pulse for electroporation.

Overview

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5

Electroporation occurs within the chamber safe, which holds the chamber rackloaded with disposable electroporation chambers; this unit is connected to the pulsecontrol by the pulse cable (figure 3). The chamber safe is designed to maintain thetemperature of the samples before, during, and after the actual electroporation. Thetemperature control compartment of the chamber safe may be filled with ice wateror water at any other desired temperature. The chamber rack has a foam rubbergasket which seals the compartment when all four slots of the chamber rack arefilled. When the lid of the chamber safe is secured with the draw latch, the entireunit can be inverted to mix the samples. The safety interlock lid protects the userfrom electrical hazard.

The samples in each of the four chambers are pulsed separately. The dot on thechamber selection knob on the top of the chamber safe (figure 4) indicates thechamber to which the pulse is directed. Between successive pulses, the pulsecontrol must be recharged and the chamber selection knob turned to the nextposition. This design allows greater flexibility because different electrical conditionsmay be selected for each sample without agitating previously pulsed samples.

Overview

Figure 4. Top View of Chamber Safe.

Draw latch

Chamber selection knob

Safety interlock lid

3

2

4

1

Cell-Porator®

Chamber Safe

™

By preparing all of the samples in disposable electroporation chambers at one timeand loading a series of chamber racks, multiple rounds of electroporation can beperformed quickly, minimizing the amount of time the cells must be held in theelectroporation buffer. This technique improves the accuracy and reproducibility ofresults. For ordering information for additional chamber racks, see Chapter 6.

Radiation-sterilized, individually packaged disposable electroporation chambers aredesigned for high transformation efficiencies with mammalian and plant cells.These chambers are made of translucent polypropylene and have leakproof caps toallow mixing of the cell suspension. Each of these chambers contains two integralflat aluminum electrodes, separated by an average distance of 0.4 cm. The tworivets through the cap of each chamber make contact with both the electrodes andthe spring-loaded contacts of the chamber safe lid when the lid is closed.

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Important: Each disposable electroporation chamber should be used only once forreliable and consistent results. Autoclaving distorts the chambers and does notremove or destroy residual DNA or other material in the crevices between chamberwalls and electrodes. Agents such as bleach that destroy DNA and other organiccompounds will damage the aluminum electrodes and alter their electricalproperties.

Warning: The chamber safe, chamber rack, and disposable electroporationchambers are for use only with the CELL-PORATOR Voltage Booster, Cat. No.11612-074 and/ or the CELL-PORATOR Pulse Control, Cat. No. 11604-055.

2.3 Overview of Electroporation

2.3.1 Background InformationElectroporation is a technique for rendering cell membranes temporarily permeableto macromolecules such as DNA by exposing cells to a brief electrical pulse orpulses of high field strength. This technique requires suspending cells between twoelectrodes to which a controlled, unidirectional electrical pulse is applied from apower supply (figure 5).

Power supply/pulse generator

Electrodes

Cell suspension

The electroporation technique originated from studies of electric field effects onbiopolymers and organelle membranes. In these studies, homogeneous electricfield pulses induced or enhanced material exchange through membranes (1–3) andalso caused membranes to fuse when in close contact with each other–aphenomenon termed electro-cell fusion (4,5).

The first successful transfer of DNA into mammalian cells by electroporation wasdescribed in 1982 (6). Since then, this method has been used successfully forintroducing foreign DNA into mammalian, plant, and microbial cells (7–14). Morerecently, other high-and low-molecular-weight molecules such as RNA (15),proteins and dyes (16), and nucleotides (17) have been introduced into cells by thismethod.

The molecular mechanism of electroporation is not well understood. It is assumedthat an electric field induces a potential difference across the membranes of thecells, exerting pressure on, and subsequently thinning, the membrane. When theinduced potential difference is increased beyond a critical point, localizedbreakdown of the membrane (“pore” formation) is thought to occur. If the fieldstrength and duration of the electric pulse is not too great, the pores close againand the cell survives; excessive field strength or pulse length, however, canirreversibly damage the membrane and lyse the cells.

Figure 5. Basic Design of an Electroporation System.

Overview

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22.3.2 Electrical Principles

Two methods can be used to apply the pulse for electroporating cells. The outputof a power supply can be used directly, applied across the sample for a length oftime determined by an electronic or mechanical switch. Alternatively, the output ofthe power supply can be used to charge a capacitor (an electrical “reservoir”) to aselected initial voltage, which is then released as a pulse of electrical currentthrough the sample; this is the method used with the CELL-PORATOR System.

Note: The two pulsing methods make different demands on power supplies andmay require different experimental conditions for optimal results.

The most important electrical parameters in an electroporation experiment are fieldstrength and pulse length. Field strength [expressed in volts per centimeter (V/cm)]is defined as the voltage applied by the power supply to the electrodes, divided bythe distance between the electrodes. The field strength is determined by the designof the power supply and the shape and dimensions of the sample chamber. Pulselength is determined by the combination of capacitance [expressed in farads (F)]and resistance [expressed in ohms (V)] in a circuit. Capacitance depends on theincorporation of capacitors in the circuit; resistance is largely determined by thesample.

In the capacitor charge pulsing method, the voltage of the pulse starts at the initialvalue and decays exponentially as a function of time, according to the followingequation:

V(t) = V0e–t/RC [1]

where V0 is the voltage at time zero, e is a constant (2.72), R is the resistance (V),and C is the capacitance (F). A plot of this function is shown in figure 6.

Overview

Figure 6. Capacitor Discharge Curve.

Time

Vol

tage

1 RC

Pulse Length

Vo

e-1 Vo

2 RC 3 RC

The pulse length of a capacitor discharge is defined as the time required for thevoltage to drop to 1/e (0.37) of its original value. The pulse length (t) can becalculated from equation [1]:

t = RC [2]

where t is expressed in seconds (s), R is expressed in V, and C is expressed in F.

In the CELL-PORATOR Pulse Control, a DC power supply is used to charge acapacitor (or group of capacitors) to the desired voltage, and the stored energy isthen discharged as a controlled electrical pulse through the disposableelectroporation chamber. The pulse control also contains a low-resistance internalresistor for discharging the capacitors or reducing the pulse length for high-resistance electroporation samples. A simplified diagram of this control circuit isshown in figure 7.

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2.3.3 Electroporation Parameters

1. Optimizing Field Strength and Pulse Length

A variety of conditions (voltages and pulse lengths) have been described forelectroporation of mammalian and plant cells. Interpretation of the results often hasbeen difficult, owing to incomplete or inconsistent descriptions of the electricalconditions, as well as divergent methods for measuring the fraction of surviving cellsand the efficiency of transient gene expression or stable transformation. Earlyresearch indicated that high field strengths (on the order of several kilovolts percentimeter) were required. However, it has since become apparent that for mostmammalian and plant cell types, maximal transient expression levels may beobtained with field strengths of 350 to 750 V/cm and pulse lengths of 1 to 10 ms(18,19).

Within limits, field strength and pulse length are complementary. Shorter pulselength can compensate for higher field strength, and vice versa. The optimal fieldstrength and pulse length for electroporation depend on the specific cell type, thecomposition of the cell suspension medium (high vs. low ionic strength), and, to alesser extent, the temperature at which electroporation is performed. Some resultsrelevant for understanding the importance of certain experimental variables arediscussed in the addendum to this manual.

2. Setting and Measuring Pulse Voltage

Pulse voltage is set with the CELL-PORATOR Pulse Control by charging the capacitorsto the desired level as indicated on the LCD voltmeter. Because the internalresistance of the apparatus is negligible, the voltage indicated by the LCD voltmeteris very close to the actual peak voltage (amplitude) of the pulse delivered to thesample.

Note: The displayed voltage (V) is not the field strength (V/cm); for disposableelectroporation chambers, which have an average distance between electrodes of0.4 cm, the field strength is 2.5 times the displayed voltage.

The most reliable way to measure the actual voltage at the disposableelectroporation chamber is to use a calibrated storage oscilloscope connected to theSCOPE outlet of the pulse control. The voltage at the connector is 0.1X the appliedvoltage.

3. Determining Pulse Length

As discussed in the section 2.3.2, the pulse length of a capacitor discharge isdependent on the capacitance and the resistance of the circuit. The resistanceincludes both the internal resistance of the wire circuit and the resistance of thedisposable electroporation chamber and its contents. This latter resistance is afunction, not only of the geometry of the chambers and the electrodes, but also ofthe composition, volume, and temperature of the cell suspension medium. Pulselengths can be determined three ways:

Figure 7. Schematic Diagram of C ELL-PORATOR System Pulse Circuit.

Electroporationchamber

DC power supply

Capacitor Internal resistorOverview

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2Determining Pulse Length by Tables. Tables 1 through 4 list approximate pulselengths for two different buffers at 0°C and 22°C across the range of capacitancesettings on the CELL-PORATOR Pulse Control. Values in these tables are accurate fordisposable electroporation chambers only, using the buffer formulations describedand with the HIGH V/LOW V Switch at the stated setting.

Note: The use of low-ionic-strength buffers may result in significant deviations fromthe values shown because of the contribution of damaged cells and added DNAsolutions to the overall conductivity.

Determining Pulse Length from Conductivity Measurements. A conductivitymeter with high internal resistance may be used to determine pulse lengths whenusing buffers, temperatures, or chambers other than those listed in tables 1 through4, or to calculate pulse times for the user's actual cell suspension. The medium orcell suspension must be measured under the same conditions (including mediumformulation, temperature, volume, and chamber) to be used when the pulse isdelivered.

Table 1. Pulse Length for HEPES-Buffered Mannitol (HBM) Solution at 0°C.

Pulse Length (ms)a

Capacitance (µF) Volumeb: 1 ml 0.75 ml 0.50 ml 0.25 ml

10 0.1 0.1 0.2 0.4

50 0.5 0.7 1.0 2.0

60 0.6 0.8 1.2 2.4

330 3.3 4.5 6.6 13.2

800 8.0 10.6 16.0 32.0

1,180 11.8 15.7 23.6 47.2

1,600 16.0 21.3 32.0 64.0

1,980 19.8 26.3 39.6 79.2

a Valid for a solution of 1 mM HEPES (pH 7), 0.55 M [10% (w/v)] mannitol when the HIGH V/LOW Vswitch is set to the HIGH V position.

b The volume of solution in each Disposable Electroporation Chamber.

Table 2. Pulse Length for HEPES-Buffered Saline (HBS) at 0°C.

Pulse Length (ms)a

Capacitance (µF) Volumeb: 1 ml 0.75 ml 0.50 ml 0.25 ml

10 0.2 0.3 0.5 0.9

50 1.2 1.5 2.3 4.6

60 1.4 1.9 2.8 5.6

330 7.7 10.2 15.3 30.7

800 18.6 24.7 37.1 74.2

1,180 27.4 36.4 54.8 109.5

1,600 37.1 49.4 74.2 148.5

1,980 45.9 61.1 91.9 183.8

a Valid for HBS buffer solution [21 mM HEPES (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4,6 mM glucose] when the HIGH V/LOW V switch is set to the LOW V position.

b The volume of solution in each Disposable Electroporation Chamber.

Overview

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Table 3. Pulse Length for HEPES-Buffered Mannitol (HBM) Solution at 22°C.

Pulse Length (ms)a

Capacitance (µF) Volumeb: 1 ml 0.75 ml 0.50 ml 0.25 ml

10 0.1 0.1 0.2 0.4

50 0.5 0.7 1.0 1.9

60 0.6 0.8 1.2 2.4

330 3.3 4.4 6.6 13.2

800 8.0 10.6 16.0 31.9

1,180 11.8 15.7 23.5 47.1

1,600 16.0 21.2 31.9 63.8

1,980 19.7 26.3 39.5 79.0

a Valid for a solution of 1 mM HEPES (pH 7), 0.55 M [10% (w/v)] mannitol when the HIGH V/LOW Vswitch is set to the HIGH V position.

b The volume of solution in each Disposable Electroporation Chamber.

Table 4. Pulse Length for HEPES-Buffered Saline (HBS) at 22°C.

Pulse Length (ms)a

Capacitance (µF) Volumeb: 1 ml 0.75 ml 0.50 ml 0.25 ml

10 0.1 0.2 0.2 0.5

50 0.6 0.7 1.1 2.2

60 0.7 0.9 1.3 2.7

330 3.7 4.9 7.4 14.8

800 9.0 11.9 17.9 35.8

1,180 13.2 17.6 26.4 52.9

1,600 17.9 23.8 35.8 71.7

1,980 22.2 29.5 44.4 88.7

a Valid for HBS buffer solution [21 mM HEPES (pH 7.05), 137 mM NaCl, 5 mM KCl,0.7 mM Na2HPO4, 6 mM glucose] when the HIGH V/LOW V switch is set to the LOW V position.

b The volume of solution in each Disposable Electroporation Chamber.

The conductivity is expressed in siemens (S = 1/V). For 1 ml of solution in adisposable electroporation chamber and the HIGH V/LOW V switch in the LOW Vposition, the pulse length is calculated by the following equation:

capacitance (F)pulse length (s) = ———————— [3]

conductivity (S)

For 1 ml of solution in a disposable electroporation chamber and theHIGH V/LOW V switch in the HIGH V position, the pulse length is calculated by thefollowing equation:

capacitance (F)pulse length (s) = —————————— [4]

0.1 + conductivity (S)

where 0.1 represents the conductivity of the internal resistor, which is introducedinto the circuit when the switch is in the HIGH V position.

Overview

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2The maximum operating current for the Chamber Safe is 10 A continuous and 40 Ainstantaneous. Damage to the Chamber Safe may result from a pulse generating acurrent of >40 A with a pulse length of >2 ms. This limit may be exceeded by theuse of a very-low-resistance buffer. When working with a buffer other than thoselisted in tables 1 through 4, the capacitance (C), voltage (V), and conductivity (S)must meet the following condition:

CV2S ¶ 32

where capacitance is measured in F, voltage in V, and conductivity in S.

Determining Pulse Length with an Oscilloscope. The use of a storageoscilloscope provides the most reliable measurement of pulse length. The pulselength is determined by measuring the time required for the voltage to decay to avalue of 0.37 (1/e) of the peak voltage.

Note: For suspension volumes other than 1 ml, the value calculated for 1 ml isdivided by the fractional volume. For example, if the pulse length for 1 ml has beendetermined to be 5 ms, the pulse length for 0.5 ml will be:

(5 ms)/0.5 = 10 ms.

4. Cell Survival Rate and Transformation Efficiency

A correlation has been observed between the fraction of cells ki l led byelectroporation and the efficiency of transient and stable transformation of thesurviving cells. This is not a simple correlation because it seems to vary widely withcell type and possibly the way cells or protoplasts are prepared for electroporation.Transient gene expression and stable transformation efficiencies are usually low iffew cells are killed. In some instances, the majority of the cells must be killed toobtain high transformation levels in the survivors. Good transformation efficiency israrely observed if less than 10% to 25% of the cells are killed. Thus, one way toobtain approximate conditions for maximum transformation is to select a pulselength between 1 and 10 ms, vary the field strength between 250 and 1,000 V/cmand determine the percentage of cells killed. In the range of 50% to 90% cell killing,field strength and pulse length can then be varied in relatively small increments todetermine optimal settings for transformation.

Very few systematic studies have been done to optimize electroporation conditionsfor stable transformation. However, where electroporation has been employed forthe generation of stable transformants, the conditions used have been similar tothose employed for efficient transient gene expression (10).

Overview

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3Operating Instructions

3.1 Procedural Overview

The basic steps of an electroporation experiment using the CELL-PORATOR

Electroporation System I are as follows:

1. Grow cells under standard conditions2. Harvest and resuspend the cells with DNA in the appropriate buffer3. Establish the parameters of electroporation4. Electroporate5. Allow the cells to take up DNA6. Determine the survival rate7. Transfer the cells into the appropriate medium8. Assay for transient gene expression 9. Select for stable transformants (if desired)

3.2 Preparing Cells for Electroporation

3.2.1 Mammalian Cells

Mammalian cells grown in culture under standard conditions are suitable forelectroporation. After harvesting, the cells should be pelleted and resuspended inthe medium of choice at a density of about 1 × 106 cells/ml. Phosphate- orHEPES-buffered saline, supplemented with various cations, have frequently beenused (7,18). Cell viability in such high-ionic-strength buffers is usually good.However, these buffers also have high conductivity, and long pulses at high voltageheat samples appreciably. Nevertheless, in most cases, these buffers are thepreferred suspension medium.

Some investigators have used weakly buffered or unbuffered mannitol or sucrosesolutions for electroporation (20–22). Cell viability in such low-ionic-strength buffersis very limited, and cells are usually kept in these solutions for the shortest timepossible. Because the conductivity of these solutions is low, higher voltages andlonger pulses can be used, but traces of culture medium (which substantiallyincrease the conductivity of the mannitol or sucrose solution) must be removed.Usually, two wash steps (centrifuging and resuspending cells in the electroporationbuffer a total of three times) are sufficient.

The conductivity of a pure mannitol or sucrose solution is very low. However, cellsuspensions in these solutions have a higher conductivity, mainly due to the saltsreleased from dead or lysed cells and from the buffer of the added DNA solution.To determine the precise pulse length in these suspensions, the conductivity of thesuspension should be measured shortly before the pulse is applied, and the pulselength should be calculated accordingly. Alternatively, an oscilloscope can be usedto record the shape and duration of the pulse. These precautions are not necessarywhen high-ionic-strength buffers are used, because the contribution by lysed cellsand the DNA solution to the overall conductivity of the suspension is negligible.

As mentioned, for most cell suspensions a density of about 1 x 106 cells/ml isappropriate. Higher cell densities may give rise to a significant proportion of electro-cell fusions. Lower cell densities decrease the yield of transformed cells but shouldnot affect transformation efficiency.

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3OperatingInstructions

3.2.2 Plant Cells

To obtain efficient transformation of plant cells, the cell walls must be digested bystandard methods (12,18,22; appendix). As with mammalian cells, differentsuspension media for plant protoplasts have been used, ranging from modifiedphosphate-buffered saline to unbuffered mannitol (12,18,22; appendix). Thesuspension media considerations described for mammalian cells apply equally toplant cells.

3.3 Setting Up the Work Area

If possible, the CELL PORATOR Electroporation System I should be set up foroperation in, or at least close to, a laminar flow hood, which allows transfer of cellsand media to and from the disposable electroporation chambers under reasonablysterile conditions. To this end, the CELL-PORATOR Electroporation System I isdesigned to be transported easily and to occupy minimal bench space.The system should be arranged so that chamber racks can be transferred betweenan ice bucket and the chamber safe without dripping water near the electronicequipment. For a sample configuration, see figure 8.

Figure 8. Operating Arrangement for the C ELL-PORATOR System.

Pulse Control with internalpower supply

Ice bucketwith Chamber Racks

Chamber Safe Oscilloscope

AC power cord

3.4 Electroporation

Before performing the following steps, review figures 1 through 4 as needed.

3.4.1 Equipment Set-Up

1. Position the components of the CELL-PORATOR Electroporation System I asdescribed in section 3.3.

2. Place the pulse control upside down on the bench, and release the spring-loaded handle lock screws. Raise the handle/stand to vertical, and lock it inplace by hand-tightening the handle lock screws. Return the unit to the raisedupright position, as shown in figure 1.Note: The main POWER switch on the rear control panel should be set toOFF.

3. Connect the pulse control unit to a storage-type oscilloscope, if desired, byplugging the coaxial cable from the oscilloscope into the SCOPE outlet on therear panel of the pulse control. Make no further electrical connections at thistime.Note: The voltage registered by the oscilloscope has to be multiplied by 10 toarrive at the actual voltage delivered in the pulse.

4. Mark the required number of culture tubes and microcentrifuge tubes. Removethe required number of disposable electroporation chambers from their plasticpouches, and if desired, place them on ice.Note: At this and all subsequent steps, take care to avoid contaminating thechambers during handling.

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5. At your option, fill the temperature control compartment of the chamber safewith ~250 ml of water at the desired temperature. Place the chamber rack in thechamber safe so that the foam rubber seal seats completely on the rim of thetemperature control compartment.

6. Open a chamber, gripping the sides of the cap. As determined by cell type,pipet 0.25 to 1 ml of a cell-DNA mixture into a 0.4-cm gap disposableelectroporation chamber or 0.20 to 0.25-µl of the cell-DNA mixture into a0.15-cm gap disposable microelectroporation chamber. Securely close the capof the chamber.

7. Place the loaded chamber in a slot of the chamber rack, and note its position(1, 2, 3, or 4). Repeat the process for each sample to be pulsed. Up to 4 samples can be placed in the chamber rack at one time.Note: Always fill all four slots of the rack with chambers, even if less than foursamples will actually be pulsed. Do not pulse any chamber that does notcontain a sample.

8. When the chamber rack is loaded with all four chambers, close the safetyinterlock lid of the chamber safe and secure it with the draw latch.

3.4.2 Electroporation

1. Plug the pulse cable into the coaxial PULSE OUTPUT connector on the back ofthe pulse control. The connection is made by sliding the connector end of thecable over the connector on the pulse control, and then twisting to lock the cordin place. Plug the double connector end of the pulse cable into the right side ofthe chamber safe.

2. If desired, resuspend the cells in 0.4-cm gap disposable electroporationchambers by inverting the chamber safe.

3. Turn the chamber selection knob on the top of the chamber safe to direct thepulse to the desired disposable electroporation chamber.

4. Verify that the main POWER switch on the rear of the pulse control is set toOFF. Plug the AC power cord (provided) into the 230 VAC INPUT receptacle,and connect to a suitable grounded 230 V power outlet.

5. Recheck all electrical connections. Turn the main POWER switch on the rearpanel of the pulse control to ON.Note: The POWER indicator light on the front panel will light up to indicate thatthe unit is connected to the line current.

6. Set the capacitor CHARGE RATE switch on the front panel of the Pulse Controlto SLOW.Note: This switch allows you to change the rate at which the capacitors arecharged and discharged when the UP and DOWN voltage control buttons arepressed.

7. Set the HIGH V/LOW V switch as needed: HIGH V when using cell suspensionbuffers of high resistance (such as mannitol solution), and LOW V when usingsuspension buffers of low resistance (such as phosphate-buffered saline).

8. Set the CAPACITANCE selector switch to the desired position. For moreinformation, review sections 2.3.2 and 2.3.3.Warning: Do not change the CAPACITANCE selector switch while thecapacitors are charged >20 V. Repeated switching with charged capacitorsmay damage the CAPACITANCE selector switch. Voltage must be drained to<20 V before changing the CAPACITANCE selector switch. Either of twomethods may be used to reduce voltage:

Method 1. Voltage control button. Press the DOWN voltage control buttonuntil the voltmeter shows <10 V. Note: This procedure may be slow if thecapacitors have been charged to a high voltage.

OperatingInstructions

15

3Operating Instructions

Method 2. Pulse discharge. Disconnect the pulse cable from the PULSEOUTPUT receptacle on the rear panel of the pulse control. Set the HIGH V/LOW V switch on the front panel to HIGH V, and turn theCHARGE/ARM switch to ARM. Push the pulse discharge TRIGGER button for1 s. This safely discharges the capacitors via the internal resistor. Followingpulse discharge, restore the previous connections and settings.

9. Set the CHARGE/ARM switch to CHARGE.10. Press the UP voltage control button and observe the LCD voltmeter. If the

voltage does not increase rapidly enough, change the CHARGE RATE switchto MED or FAST, and continue the procedure. Press the UP button until thevoltage reading is 5 to 10 V higher than the desired discharge voltage.Note : Once the capacitors have been charged, a slow discharge will beobserved, due to the internal resistance of the voltmeter. The rate of this drainis not affected by the CHARGE RATE setting. The precise amount ofovercharge needed will vary, depending on the CAPACITANCE setting chosen.The voltage delivered to the chamber is the voltage displayed at the momentthe pulse discharge TRIGGER button is depressed.

11. If the capacitors have been charged too high, press the DOWN voltage controlbutton until the desired voltage is reached. Adjust the CHARGE RATE switchas needed to facilitate reaching the desired voltage reading.Warning: The maximum operating current for the chamber safe is 10 A continuous and 40 A instantaneous. Damage to the chamber safe mayresult from a pulse generating a current of >40 A with a pulse length of >2 ms.This limit may be exceeded by the use of a very low resistance buffer. Thecapacitance (C), voltage (V), and conductivity (S) must meet the followingcondition:

CV2S ¶ 32

where capacitance is in F, voltage in V, and conductivity in S. For moreinformation, review section 2.3.3.

12. When ready to deliver the electric pulse to the disposable electroporationchamber, set the CHARGE/ARM switch to ARM.Note: The ARM indicator light on the front panel will light up to indicate that theunit is ready to deliver a pulse.IMPORTANT! Before pulsing, double check all connections and settings:• HIGH V/LOW V switch set as desired• CAPACITANCE at selected setting• LCD voltmeter showing desired voltage • Cables attached properly• Chamber selection knob directed to desired chamber

13. Depress the pulse discharge TRIGGER button, and hold for 1 s.Note: The LCD voltmeter should now read <20 V. If a reading of >20 V isobserved, discharge the capacitors using Method 2 as described in step 8, andrefer to Chapter 4 for troubleshooting suggestions (“The LCD voltmeter doesnot show a decrease in voltage when the pulse discharge TRIGGER button isdepressed”).

14. For additional samples, change the chamber selection knob on the chambersafe to the next desired position, and repeat steps 6 through 13 until all of thesamples have been pulsed.

15. Once the experiment is finished, check the voltmeter to make sure thecapacitors are discharged (the voltage display should read <20 V).

16. Turn the main POWER switch on the rear panel of the pulse control to OFF.Turn off all other instruments. Disconnect the pulse control from the wall outletfirst, then from all other electrical components.

16

3.4.3 Post-Electroporation

1. Allow the cells to rest for a few minutes for uptake of DNA.2. Open the lid of the chamber safe, and remove the chamber rack and chambers.

Remove an aliquot of cells for staining to determine the percent of survivors.3. Transfer the cells into culture dishes containing the appropriate medium, and

incubate.4. Harvest the cells, or an aliquot of them, and assay for transient gene

expression.Note: Common assay methods measure the expression of a marker gene suchas chloramphenicol acetyltransferase (CAT), b-galactosidase, or luciferase. Foradditional information on this and the preceding steps, see the Appendix.

5. Transfer the cells into a selective medium to select for stable transformants, ifdesired.

6. Use a damp cloth or paper towel to wipe off any spills from the instruments.Store the components in a dry, noncorrosive environment.

OperatingInstructions

17

4Troubleshooting Guide

Many apparatus problems can be solved by carefully following the instructions in this manual. Some suggestions fortroubleshooting are given in this chapter. Should these suggestions not resolve the problem, call the TECH-LINESM at one ofthe numbers listed on the back cover of this manual. If the unit must be returned for repair, contact the Customer ServiceDepartment or your local distributor for shipping instructions. Include a full description of the problem.

4.1 General Troubleshooting Guidelines

Problem Comment

The POWER indicator light does not come on Check that the AC power cord is securely connected to the AC power input on the rear panel. If the LCD voltmeter is functional and the problem persists, the POWER indicator light bulb is burned out.

Check the line fuse and replace if blown.

The ARM indicator light does not come on Check that the CHARGE/ARM switch is in the ARM position.

Check that the AC power cord is securely connected to the AC power input on the rear panel. If the LCD voltmeter is functional and the problem persists, the ARM indicator light bulb is burned out.

The pulse indicator light does not flash when a pulse is The pulse indicator light bulb is burned out.discharged; voltage drops on the LCD voltmeter display

The LCD voltmeter shows no display Check that the AC power cord is securely connected to the AC power input on the rear panel.

Check the line fuse and replace if blown.

The LCD voltmeter does not show any change in voltage Verify that the CHARGE/ARM switch is set to CHARGE.when either the UP or the DOWN voltage control button isdepressed

Water leaks from the temperature control compartment when Make sure all four positions of the chamber rack are filled.the chamber safe is inverted

Adjust the draw latch, rotating it counterclockwise one full turn at a time, until the leaking stops. Note: Some units are adjusted by rotating the metallic collar on the draw latch.

The LCD voltmeter does not show a decrease in voltage when Verify that the CHARGE/ARM switch is set to ARM.the pulse discharge TRIGGER button is depressed; the pulse indicator light may remain lit Check the electrical connections between the chamber safe and the

pulse control.

Check that the chamber safe lid is securely locked.

Verify that the chamber selection knob is set to the desired position, and that the slot in that position contains a disposable electro-poration chamber.

Check for the presence of cell suspension solution in the chamber.

Rotate the chamber selection knob two or three times to remove any oxidation that may be present on switch contacts.

If the problem is still not resolved, necessitating further attention or repair, the pulse control unit must first be discharged, using either method described in step 8 of the “Electroporation” protocol.

18

4.2 Pulse Control Checkout

Perform the following test sequence for a routine inspection or diagnostic evaluationof the pulse control with internal power supply. You will need an oscilloscope toperform the complete procedure. These steps should be performed withoutconnecting the pulse control to the chamber safe. Review figure 2, as needed, tofamiliarize yourself with the front and rear control panels.

4.2.1 Basic Controls1. Connect the pulse control to the AC power outlet.2. Set the CHARGE/ARM switch to CHARGE.3. Set the CAPACITANCE switch to 10 µF.4. Set the HIGH V/LOW V switch to LOW V.5. Turn the POWER switch located on the rear of the unit to ON.

• The POWER indicator light should now be lit.• The LCD voltmeter should show a reading.

6. Press the UP voltage control button.• The LCD voltmeter reading should increase.

7. Press the DOWN voltage control button.• The LCD voltmeter reading should decrease.

8. Repeat steps 6 and 7 for each setting of the CHARGE RATE switch.

4.2.2 Pulse Discharge Functions1. With the CHARGE RATE switch set at MED, press the UP voltage control

button until the LCD voltmeter indicates between 100 and 200 V.2. Switch the CHARGE/ARM switch to ARM.

• The ARM indicator light should now be lit.3. Press the pulse discharge TRIGGER button, and hold for at least 1 s.

• The pulse indicator light should flash if the voltage is Ä25 V.4. Set the HIGH V/LOW V switch to HIGH V.5. Set the CHARGE/ARM switch to CHARGE, and repeat steps 1 and 2.6. Press the pulse discharge TRIGGER button, and hold for at least 1 s.

• The pulse indicator light should flash briefly.• The LCD voltmeter reading should drop to near 0 V.

4.2.3 Oscilloscopic Analysis1. Set the CHARGE/ARM switch to CHARGE.2. Press the UP voltage control button until the LCD voltmeter indicates between

100 and 200 V.3. Connect an oscilloscope to the coaxial SCOPE outlet on the rear panel.4. Press the pulse discharge TRIGGER button, and hold for at least 1 s.

• The pulse indicator light should not flash.• The oscilloscope should not indicate an output pulse.

5. Switch the CHARGE/ARM switch to ARM.6. Press the pulse discharge TRIGGER button, and hold for at least 1 s.

• The pulse indicator light should flash.• The oscilloscope should indicate a pulse of 0.1 ms.

Note: The pulse length is the time necessary for the voltage to drop to 1/e(0.37) of its peak value. The SCOPE outlet uses a 10:1 divider to reducethe pulse amplitude delivered to the oscilloscope input.

TroubleshootingGuide

19

4TroubleshootingGuide

7. Repeat steps 1 through 6 for each position of the CAPACITANCE switch.• Pulse lengths will increase with increasing capacitance.

8. Disconnect the oscilloscope from the SCOPE outlet. Connect an oscilloscopewith a suitable voltage divider network or 100X probe to the coaxial PULSEOUTPUT connector on the rear panel.

9. Set the CHARGE/ARM switch to CHARGE.10. Press the UP voltage control button until the LCD voltmeter indicates between

100 and 200 V.11. Set the HIGH V/LOW V switch to LOW V.12. Set the CHARGE/ARM switch to ARM.13. Press the pulse discharge TRIGGER button, and hold for at least 1 s.

• The oscilloscope should indicate a value equal to the charging voltage onthe LCD voltmeter in step 10 (±10%).

14. Set the HIGH V/LOW V switch to HIGH V.15. Press the pulse discharge TRIGGER button, and hold for at least 1 s to

discharge the capacitor(s).• The LCD voltmeter should read <20 V.

16. Turn the POWER switch on the rear panel to OFF, and unplug the unit from theAC power source. Disconnect the oscilloscope.

20

5References and Select Bibliography

5.1 References1. Neumann, E. and Katachalsky, A. (1972) Proc. Natl. Acad. Sci. USA 69, 993.2. Neumann, E. and Rosenheck, K. (1972) J. Membr. Biol. 14, 194.3. Zimmermann, U. (1982) Biochim. Biophys. Acta 694, 227.4. Scheurich, P., Zimmermann, U., Mischel, M., and Lamprecht, I. (1980)

Z. Naturforsch. Teil C Biochem. Biophys. Biol. Virol. 37, 1081.5. Neumann, E., Gerisch, G., and Opatz, K. (1980) Naturwissenschaften 67, 414.6. Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P.H. (1982)

EMBO J. 1, 841.7. Potter, H., Weir, L., and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161.8. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati,

R.S. (1985) Nature (Lond.) 317, 230.9. Reiss, M., Jastreboff, M.M., Bertino, J.R., and Narayanan, R. (1986) Biochem.

Biophys. Res. Commun. 137, 244.10. Toneguzzo, F., Hayday, A.C., and Keating, A. (1986) Mol. Cell. Biol. 6, 703.11. Tur-Kaspa, R., Teicher, L., Levine, B.J., Skoultchi, A.I., and Shafritz, D.A.,

(1986) Mol. Cell. Biol. 6, 716.12. Fromm, M., Taylor, L.P., and Walbot, V. (1986) Nature (Lond.) 319, 791.13. Bates, G., (1986) unpublished observations.14. Nishiguchi, M., Langridge, W.H.R., Szalay, A.A., and Zaitlin, M. (1986) Plant

Cell Rep. 5, 57.15. Okada, K., Nagata, T., and Takebe, I. (1986) Plant Cell Physiol. 27, 619.16. Sowers, A. S. and Lieber, M. R. (1986) FEBS Lett. 205, 179.17. Sokoloski, J.A., Jastreboff, M.M., Bertino, J.R., Sartorelli, A.C., and Narayanan,

R. (1986) Anal. Biochem. 158, 272.18. Fromm, M., Taylor, L. P., and Walbot, V. (1985) Proc. Natl. Acad. Sci. USA 82,

5824.19. Rabussay, D., Uher, L., Bates, G., and Piastuch, W. (1987) FOCUS® 9:3, 1.20. Shimizu, H. and Watanabe, T. (1986) Med. Immunol. 11, 105.21. Glassy, M.C., unpublished observations.22. Riggs, C.D. and Bates, G.W. (1986) Proc. Natl. Acad. Sci. USA 83, 5602.

5.2 Select BibliographyBooks and Review Articles

Fromm, M., Callis, J., Taylor, L.P., and Walbot, V. (1987) Methods Enzymol.153, 351.Knight, D. E. and Scrutton, M. C. (1986) Biochem. J. 234, 497.Neumann, E. and Bierth, P. (1986) Am. Biotechnol. Lab. 4, 10.Zimmermann, U. (1982) Biochim. Biophys. Acta 694, 227.

Mammalian CellsBoggs, S.S., Gregg, R.G., Borenstein, N., and Smithies, O. (1986) Exp.Hematol. (N.Y.) 14, 988.Chu, G., Hayakawa, H., and Berg, P. (1987) Nucleic Acids Res. 15, 1311.

21

5Evans, G. A., Ingraham, H.A., Lewis, K., Cunningham, K., Seki, T., Moriuchi, T.,Chang, H.C., Silver, J., and Hymann, R. (1984) Proc. Natl. Acad. Sci. USA 81,5532.Falkner, F.G. and Zachau, H.G. (1984) Nature (Lond.) 310, 71.Gordon, P.B., Hoyvik, H., and Seglen, P.O. (1985) Prog. Clin. Biol. Res. 180,475.Gordon, P.B. and Seglen, P.O. (1982) Exp. Cell Res. 142, 1.Hama-Inaba, H., Shiomi, T., Sato, K., Ito, A., and Kasai, M. (1986) Cell Struct.Funct. 11, 191.Kinosita, K. and Tsong, T.Y. (1977) Nature (Lond.) 268, 438.Muhlig, P., Forster, W., Jacob, H. E., Berg, H. Roglin, G., and Wohlrabe, K.(1984) Stud. Biophys. 104, 207.Narayanan, R., Jastreboff, M.M., Chiu, C.F., and Bertino, J.R. (1986) Biochem.Biophys. Res. Commun. 141, 1018.Neumann, E. (1984) Bioelectrochem. Bioenerg. 13, 219.Neumann, E., Schaefer-Ridder, M., Wang Y., and Hofschneider, P.H. (1982)EMBO J. 1, 841.Potter, H., Weir, L., and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161.Rabussay, D., Uher, L., Bates, G., and Piastuch, W. (1987) FOCUS 9:3, 1.Reiss, M., Jastreboff, M. M., Bertino, J.R., and Narayanan, R. (1986) Biochem.Biophys. Res. Commun. 137, 244.Shimizu, H. and Watanabe, T. (1986) (In Japanese) Med. Immunol. 11, 105.Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapati, R.S. (1985) Nature (Lond.) 317, 230.Sokoloski, J.A., Jastreboff, M.M., Bertino, J.R. Sartorelli, A.C., and Narayanan,R. (1986) Anal. Biochem. 158, 272.Sowers, A.E. (1986) J. Cell Biol. 102, 1358.Sowers, A.E. and Lieber, M.R. (1986) FEBS Lett. 205, 179.Stopper, H., Zimmermann, U., and Wecker, E. (1985) Z. Naturforsch Sect. CBiosci. 40, 929.Tolleshaug, H., Gordon, P.B., Solheim, A.E., and Seglen, P.O. (1984) Biochem.Biophys. Res. Commun. 119, 955.Toneguzzo, F., Hayday, A. C., and Keating, A. (1986) Mol. Cell. Biol. 6, 703.Toneguzzo, F. and Keating, A. (1986) Proc. Natl. Acad. Sci. USA 83, 3496.Tur-Kaspa, R., Teicher, L., Levine, B.J., Skoultchi, A.I., and Shafritz, D.A.(1986) Mol. Cell. Biol. 6, 716.Wong, T.-K. and Neumann, E. (1982) Biochem. Biophys. Res. Commun. 107,584.Zerbib, D., Amalric, F., and Teissie, J. (1985) Biochem. Biophys. Res.Commun. 129, 611.

Plant CellsBoston, R.S., Becwar, M.R., Ryan, R.D., Goldsbrough, P.B., Larkins, B.A., andHodges, T.K. (1987) Plant Physiol. (Bethesda) 83, 742.Cutler, A.J. and Saleem M. (1987) Plant Physiol. (Bethesda) 83, 24.Ecker, J.R. and Davis, R.W. (1986) Proc. Natl. Acad. Sci. USA 83, 5372.Fromm, M., Taylor, L.P., and Walbot, V. (1985) Proc. Natl. Acad. Sci. USA 82,5824.Fromm, M., Taylor, L.P., and Walbot, V. (1986) Nature (Lond.) 319, 791.Koncz, C., Olsson, O., Langridge, W.H.R., Schell, J., and Szalay, A.A. (1987)Proc. Natl. Acad. Sci. USA 84, 131.

References andSelect Bibliography

22

Langridge, W.H.R., Li, B.J., and Szalay, A.A. (1985) Plant Cell Rep. 4, 355.Mehrle, W., Zimmermann, U., and Hampp, R. (1985) FEBS Lett. 185, 89.Morikawa, H., Iida, A., Matsui, C., Ikegami, M., and Yamada, Y. (1986) Gene41, 121.Neumann, E. (1984) Bioelectrochem. Bioenerg. 13, 219.Nishiguchi, M., Langridge, W. H.R., Szalay, A.A., and Zaitlin, M. (1986) PlantCell Rep. 5, 57.Okada, K., Nagata, T., and Takebe, I. (1986) Plant Cell Physiol. 27, 619.Ou-Lee, T.M., Turgeon, R., and Wu, R. (1986) Proc. Natl. Acad. Sci. USA 83,6815.Ow, D. W., Wood, K.V., DeLuca, M., De Wet, J.R., Helinski, D.R., and Howell,S.H. (1986) Science 234, 856.Perani, L., Radke, S., Wilke-Douglas, M., and Bossert, M. (1986) Physiol. Plant.68, 566.Rabussay, D., Uher, L., Bates, G., and Piastuch, W. (1987) FOCUS 9:3, 1.Riggs, C.D. and Bates, G. W. (1986) Proc. Natl. Acad. Sci. USA 83, 5602.Shillito, R.D., Saul, M.W., Paszkowski, J., Muller, M., and Potrykus, I. (1985)BioTechnology 3, 1099.Thornburg, R.W., An, G., Cleveland, T.E., Johnson, R., and Ryan, C.A. (1987)Proc. Natl. Acad. Sci. USA 84, 744.Yang, N. S. (1985) Trends Biotechnol. 3, 191.Zachrisson, A. and Bornman, C.H. (1986) Physiol. Plant. 67, 507.

Microbial CellsHashimoto, H., Morikawa, H., Yamada, Y., and Kimura, A. (1985) Appl.Microbiol. Biotechnol. 21, 336.Lian-ying, L.Y. and Wong, T. (1984) Antibiot. 9, 450.Shivarova, N., Forster, W., Jacob, H.-E., and Grigorova, R. (1983) Mikrobiol.23, 595.

References andSelect Bibliography

23

Product Size Cat. No.

Accessories:Pulse Control each 11604-055

Chamber Safe 4-chamber capacity 11602-018

Chamber Rack each 11606-019

0.4-cm gap Disposable Electroporation Electroporation Chambers pkg. of 36 11601-010

pkg. of 50 11601-028

0.15-cm gap Disposable Microelectroporation Chambers pkg. of 36 11608-015

pkg. of 50 11608-031

CELL-PORATOR Voltage Booster each 11612-074

6Related Products

24

7Additional Information

7.1 Care and HandlingThe components of the CELL-PORATOR Electroporation System I are fabricated from ABS,aluminum, acrylic, and silicone. As with any laboratory instrument, adequate careensures consistent and reliable performance. The external surfaces of the componentsmay be cleaned with a damp cloth or paper towel and mild detergent. To remove greaseand oils, use a light application of hexane, kerosene, or aliphatic naphtha. Never useabrasive cleaners, window sprays, or rough cloths to clean the components, as thesecan cause surface damage.

Additional cautions:• Do not submerge the apparatus in any liquid.• Do not allow any electrical parts to come into direct contact with liquid.• Do not autoclave or dry-heat sterilize the apparatus.• Do not expose the apparatus to phenol, acetone, benzene, halogenated

hydrocarbon solvents, undiluted laboratory alcohols, or any corrosive chemicals.• Avoid prolonged exposure of the apparatus to UV light.• Do not expose the apparatus to heat, moisture, or excessive dust.

Important: Each disposable electroporation chamber should be used only once forreliable and consistent results, and should be disposed of properly following use.Autoclaving distorts the chambers and does not remove or destroy residual DNA or othermaterial in the crevices between chamber walls and electrodes. Agents such as bleachthat destroy DNA and other organic compounds will damage the aluminum electrodesand alter their electrical properties.

7.2 Maintenance

The following simple, routine inspection and maintenance procedures will help ensureboth the safety and the performance of the CELL-PORATOR System.

1. Inspect all electrical connections regularly. If the connectors to either the pulsecontrol or the chamber safe do not appear to be in proper working condition,replace the pulse cable or call the TECH-LINE.

2. If the pulse cable shows any signs of wear or damage (fraying, cracking, nicks,abrasions, or melted insulation), replace it immediately.

3. Do not open the instrument case of any CELL-PORATOR System component.These apparatus contain no user-serviceable parts. If you suspect internalproblems with any component of this system, call the TECH-LINE immediately.

7.3 SpecificationsPulse Control with Internal Power SupplyWeight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .(4.4 kg) 9.8 lbDimensions (W x L x H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16.5 x 25.4 x 14.0 cm (6.5 x 10.0 x 5.5 in.)Case materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ABS, aluminumInsulation rating for SCOPE connected cable . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .400 V (minimum)Installation category . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Type 2Operating temperature range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0°C to 30°CHumidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .noncondensing atmospherePulse cord rating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5,000 VPower requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 VAC, 50/60 Hz, 45 WCapacitance values (µF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10, 50, 60, 330, 800, 1180, 1600, 1980Capacitor charge rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Low, medium, highInternal resistor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 V, parallel to chamberOscilloscope output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .BNC, 0.1X actualPulse output receptacle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SHV Receptacle Kings P.N. 1704-1Maximum pulse current output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 AVoltmeter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Digital LCD, ±5% accuracy

25

7Main fuse rating:220 ± 20 Vrms operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .T0.5 A/250

Chamber SafeWeight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.0 kg (2.2 lb)Dimensions (W x L x H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17.8 x 17.8 x 10.2 cm (7.0 x 7.0 x 4.0 in.)Temperature control compartment volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250 mlConstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ABS, acrylicMaximum operating current . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 A continuous and 40 A instantaneousPulse cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Custom molded dual banana jack, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .SHV Plug Kings #1705-2, 30" long, coaxial cable

Chamber RackWeight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100 g (0.2 lb)Dimensions (W x L x H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11.7 x 10.4 x 4.6 cm (4.6 x 4.1 x 1.8 in.)Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acrylic, siliconeDisposable Electroporation ChamberOverall dimensions (W x L x H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 x 4.8 x 3.0 cm (1.7 x 1.9 x 1.2 in.)Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polypropylene, aluminumWorking volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25 to 1 mlAverage electrode gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 cm

7.4 Warranty

Life Technologies Inc. warrants apparatus of its manufacture against defects in materialsand workmanship, under normal service, for one year from the date of receipt by thepurchaser. This warranty excludes damages resulting from shipping, misuse,carelessness or neglect. Life Technologies Inc. liability under the warranty is limited tothe repair of such defects or the replacement of the product, at its option, and is subjectto receipt of reasonable proof by the customer that the defect is embraced within theterms of the warranty. All claims made under this warranty must be presented to LifeTechnologies Inc. within one year following the date of delivery of the product to thecustomer.

This warranty is in lieu of any other warranties or guarantees, expressed orimplied, arising by law or otherwise. Life Technologies Inc. makes no otherwarranty, expressed or implied, including warranties of merchantability or fitnessfor a particular purpose. Under no circumstances shall Life Technologies Inc. beliable for damages either consequential, compensatory, incidental or special,sounding in negligence, strict liability, breach of warranty or any other theory,arising out of the use of the product listed herein.

Life Technologies reserves the right to make improvements in design, construction, andappearance without notice.

7.5 Declaration of Conformity and CE Mark

Note: The information outlined in this section applies only to customers located in theEuropean Union (EU). The EU is currently comprised of 15 member countries.

This laboratory apparatus is identified with the CE mark. This mark indicates that theproduct complies to the following EU Directives and Standards:

Application of Council Directive(s):73/23/EEC Low Voltage Directive89/336/EEC Electromagnetic Compatibility

Standards:EN 61010-1:1993 Product SafetyEN 50081-1:1992 EmissionsEN 50082-1:1992 Immunity

EU Representative:Life Technologies Ltd.

EU Address:3 Fountain Dr.Inchinnan Business ParkPaisley, PA49RF Scotland

A copy of the Declaration of Conformity certificate is available upon request.

AdditionalInformation

26

Appendix: Experimental DataAElectroporation is an emerging technology. As such, the parameters that affect thetransfer of DNA into electroporated cells and the fate of the transferred DNA are stillbeing investigated. Scientists and consultants of Life Technologies, Inc. haveperformed a series of experiments to define conditions for effective electroporationof some mammalian and plant cells. The results of these experiments are presentedhere as examples for designing and executing electroporation experiments withvarious cell types.

A.1 Electroporation of Mammalian Cells

Experiments were performed with COS-7 cells (green monkey kidney). HEPES-buffered saline (HBS; high ionic strength, low resistance) or HEPES-bufferedmannitol (HBM; low ionic strength, high resistance) was used as the suspensionbuffer for electroporation. The influence of field strength, pulse length, ionicstrength, temperature, DNA concentration, DNA conformation, presence of carriernucleic acid, and time of incubation in the DNA solution after electroporation on thelevel of transient gene expression and cell killing were studied.

A.1.1 Materials and Methods• Material. GIBCO BRL media reagents and plasticware for cell culture; Sigma

Acetyl Coenzyme A and salmon sperm DNA; and New England Nuclear D-Threo-[Dichloroacetyl-1, 2-14C]-Chloramphenicol (14C-CAM; 60 Ci/mol)were used in these experiments.

• Plasmid DNA. Plasmid pSV2CAT (Gorman et al., 1982) was isolated from E. coli HB101 after amplification with CAM. Plasmid DNA was purified by thealkali/SDS method (Maniatis et al., 1982) and stored in 10 mM Tris-HCl about90% supercoiled molecules; the remainder were mostly relaxed circularmolecules. Plasmid pSV2CAT contained the CAT gene whose expressionwas measured after electroporation to determine the extent of transient geneexpression.

• Cell growth. COS-7 cells were grown in Dulbecco’s modification of Eagle’smedium (D-MEM) supplemented with 100 units/ml penicillin, 0.1 mg/mlstreptomycin, 2 mM glutamine and 10% heat-inactivated fetal bovine serum at37°C, 10% CO2, to half-confluence (1.2 x 105 cells/cm2). Cells were harvestedby the trypsin/EDTA procedure, washed once in HBS [21 mM HEPES (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose] and resuspended in the appropriate buffer for electroporationat a density of 6.25 x 106 cells/ml.

• Electroporation. Unless noted otherwise, the following standard procedurewas used. A 0.8-ml aliquot of the cell suspension (5 x 106 cells) in either HBS(high ionic strength buffer) or HBM [low ionic strength buffer containing 1 mM HEPES (pH 7.0) and 0.55 M (10% w/v) mannitol] was pipetted into theelectroporation chamber which already contained 500 µg carrier DNA(sonicated, heat-denatured salmon sperm DNA) and 10 µg plasmid DNA in0.2 ml of TE buffer [20 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA]. After gentlymixing the DNA and cells by inverting the electroporation chamber, thesuspension was chilled in ice water. The electroporation chambers wereplaced into the chamber safe and, if necessary, the cells were resuspendedshortly before applying the electrical pulse by gently inverting the chamber

27

Asafe. The chamber safe was connected to the pulse control unit with internalpower supply. After setting the desired capacitance and voltage, each samplewas pulsed once. The electroporation chambers were removed from thechamber safe and kept chilled in ice water for 10 min.

• Treatment of cells after electroporation. After recovering the cellsuspension from the electroporation chambers with a sterile 1-ml pipette, 0.7 ml of the suspension was mixed with 14 ml of supplemented D-MEM andincubated at 37°C in cell culture flasks under 10% CO2 for 68 to 72 h.

• Viability tests. The Trypan Blue exclusion test was used for determining theviability of mammalian cells at 10 min after electroporation unless notedotherwise.

• Preparation of cell extracts (Gorman et al., 1982). After 68 to 72 h, cellswere harvested by the trypsin/EDTA method, washed once with HBS andresuspended in 100 µl of 0.25 M Tris-HCl (pH 7.5), 0.1 mM phenyl-methylsulfonyl fluoride (PMSF) in a 15-ml conical tube. At this point cells wereeither stored frozen or lysed immediately by sonication. The cell homogenate(about 150 µl) was incubated at 65°C for 10 min and transferred to a 1.5-ml microcentrifuge tube. After centrifugation, about 250 µl of supernatant(sample extract) was recovered and assayed for CAT activity or stored at –20°C.

• Assay for Chloramphenicol Acetyl Transferase (CAT) (Gorman et al.,1982). A 20-µl aliquot of sample extract was added to 25 µl 1 M Tris-HCl(pH 7.5), 2 µl (0.2 µCi) 14C-CAM and 1.5 µl 40 mM Acetyl Coenzyme A. Thevolume was adjusted to 150 µl with water and the mixture incubated for 2 h at37°C. The reaction mixture was extracted two times with 150 µl of ethylacetate. The combined organic phases were evaporated either by heating theliquid in an open microcentrifuge tube in a heating block at 60°C for 1 h or byletting it sit in a fume hood overnight. The dried CAM was dissolved in 20 µl ethyl acetate, 5 µl was spotted on a thin layer chromatography (TLC)plate (Bakerflex Silica Gel 1B-F) and the plate was chromatographed in a TLCchamber using chloroform:methanol (95:5, v/v) as the solvent. After thesolvent front had migrated about 10 cm, the plate was dried andautoradiographed overnight at room temperature with Kodak XAR-5 film. Thepositions of radioactive spots on the TLC plate were marked, each samplelane was cut out, and each strip was cut into five pieces containing thesample origin, chloramphenicol, 1-acetylchloramphenicol, 3-acetyl-chloramphenicol and 1,3-diacetylchloramphenicol, respectively. Each piecewas counted separately in a liquid scintillation counter.The CAT activity is expressed as percent of 14C-CAM converted to acetylatedforms using the formula:

% conversion of 14C-CAM = cpm in acetylated spots x 100/total cpm

Note: CAT activities above 60% conversion of 14C-CAM are beyond thelinear range of the assay and therefore are underestimates of the true level ofactivity.

Experimental Data

28

A.1.2 Results and Observations

Experiment 1: Transient gene expression and cell survival as a function of pulse lengthand field strength I (figures A-1 and A-2)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (5 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .HBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . .500 or 750 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200 or 300 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to 37.1 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Experimental Data

00

20

40

60

80

100

10 20 30 40

Pulse Length (ms)

Per

cent

Figure A-1. Transient Gene Expression and Cell Survival as a Function of Pulse Lengthat 500 V/cm. Percent conversion of 14C-CAM (O); Percent viable cells (D).

00

20

40

60

80

100

10 20 30 40

Pulse Length (ms)

Per

cent

Figure A-2. Transient Gene Expression and Cell Survival as a Function of Pulse Lengthat 750 V/cm. Percent conversion of 14C-CAM (O); Percent viable cells (D).

Observations:1. Killing and CAT expression are relatively low at 500 V/cm at all pulse lengths

tested, although CAT expression is significantly higher at pulse lengths >2 ms.

2. At 750 V/cm, high transient gene expression is observed with pulse lengthsÄ7.7 ms. In this experiment the highest level of transient gene expression isobserved when 80% or more of the cells are killed during electroporation.

29

AExperiment 2: Transient gene expression and cell survival as a function of pulse lengthand field strength II (figure A-3)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (5 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .HBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . .250 or 1,000 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100 to 400 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to 27.5 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Experimental Data

00

20

40

60

80

100

10 20 30

Per

cent

Con

vers

ion

of 14

C-C

AM

1

2

4

3

Panel A

Panel B

00

20

40

60

80

100

10 20 30

Pulse Length (ms)

Per

cent

Cel

l Sur

viva

l

1

4

3

1

2

Figure A-3. Transient Gene Expression and Cell Survival as a Function of Pulse Lengthand Field Strength. Panel A: Cat expression [percent conversion of 14C-CAM (O)]. Panel B: Cell survival [percent viable cells (D)]. Curves 1,2,3 and 4 correspond to fieldstrengths of 250, 500, 750, and 1,000 V/cm, respectively.

Observations:1. An increase in field strength and pulse length generally increases transient

gene expression and decreases the percentage of surviving cells.2. At a field strength of 250 V/cm almost no transient gene expression is

observed. In this set of experiments the highest level of transient geneexpression is obtained at a field strength of 1,000 V/cm and a pulse length ofabout 10 ms.

30

Experiment 3: Transient gene expression and cell survival as a function of thecomposition of the electroporationbuffer at different field strengths (figure A-4)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . .COS-7 (3.6 x 106 cells/ml in HBS and

2.9 x 106 cells/ml in HBM)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . .HBS or HBMfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . .250 to 1000 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100 to 400 VV setting . . . . . . . . . . . . . . . . . .LOW V for HBS; HIGH V for HBMpulse length . . . . . . . . . . . . . . . . . . .7.1 ms in HBS; 8.0 ms in HBMtemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Experimental Data

00

20

40

60

80

100

250 500 750 1000

Per

cent

Panel A

00

20

40

60

80

100

250 500 750 1000

Field Strength (V/cm)

Per

cent

Panel B

Figure A-4. Transient Gene Expression and Cell Survival as a Function of theComposition of the Electroporation Buffer at Different Field Strengths. Panel A: Percentconversion of 14C-CAM (O) and percent viable cells (D) in HBS. Panel B: Percent conversionof 14C-CAM (O) and percent viable cells (D) in HMB.

Observations:1. Electroporation in HBS: The number of surviving cells is inversely related to

the efficiency of transient gene expression up to about 625 V/cm. At this fieldstrength maximal CAT activity is reached under these experimentalconditions. Higher field strengths up to 1,000 V/cm do not significantlychange the percentage of survivors or the level of CAT activity.

2. Electroporation in HBM: The cell survival curve is similar to that observed inHBS. However, CAT expression reaches a maximum at about 625 V/cm anddeclines significantly at higher field strengths. Maximal CAT expression ofcells electroporated in HBM is less than half of that observed for cellselectroporated in HBS.

31

AExperiment 4: Transient gene expression and cell survival after electroporation in HBSand D-MEM (table A-1)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (5 x 106 cells/ml)electroporation buffer . . . . . . . . . . .HBS or D-MEM (high glucose)field strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .500 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15.5 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Cells were harvested as described in section A.1.1, resuspended once in HBS,centrifuged and resuspended in D-MEM (high glucose); these steps were performedat room temperature. All other procedures were performed as described in sectionA.1.1. The values given in table A-1 are the average of duplicates obtained in thesame experiment.

Table A-1. CAT Expression and Cell Survival After Electroporation in HBS or D-MEM

% Conversion of 14C-CAM % Viable Cells

HBS 56 6 2 30 6 8

D-MEM 81 610 54 6 0

Observation:Both the level of transient gene expression and the proportion of surviving cells aregreater if electroporation is performed in D-MEM rather than in HBS.

Experimental Data

32

Experiment 5: Transient gene expression and cell survival as a function of temperatureat different pulse lengths and ionic strengths (figure A-5)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (2.7 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . .HBS or HBMfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .375 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150 VV setting . . . . . . . . . . . . . . . . . .LOW V for HBS; HIGH V for HBMpulse length . . . . . . . . . .0 to 27.4 ms at 2°C; 0 to 13.1 ms at 22°C

(measured with an oscilloscope)temperature at electroporation . . . . . . . . . . . . . .0 to 4°C and 22°C

The experiment was performed as described in section A.1.1, except that a portionof the cells were kept at room temperature (22°C) before, during, and afterelectroporation.

Experimental Data

00

20

40

60

80

100

10 20 30

Pulse Length (ms)

Per

cent

Panel B: 22°C

00

20

40

60

80

100

10 20 30

Per

cent

Panel A: 2°C

Figure A-5. Transient Gene Expression and Cell Survival as a Function of Temperatureat Different Pulse Lengths in HBS and HBM. Panel A: 2°C. Panel B: 22°C. Percentconversion of 14C-CAM after electroporation in HBS (O) and HBM (), respectively; percentviable cells after electroporation in HBS (D) and HBM (), respectively.

Observations:1. Transient gene expression in COS-7 cells is higher when electroporation is

performed at room temperature rather than at 0 to 4°C.2. Transient gene expression is much higher when electroporation is performed

in HBS compared to electroporation in HBM.3. An increase in pulse length up to about 10 ms results in increased transient

gene expression. Pulse lengths greater than 10 ms do not yield significantlyhigher levels of gene expression or might even decrease it.

4. Maximal transient gene expression under these experimental conditions isobtained at room temperature at a pulse length of about 10 ms in HBS.

33

AExperiment 6: Transient gene expression and cell survival as a function of the time cellsare kept in the electroporation buffer after electroporation (figure A-6)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (5 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .HBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .500 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18.6 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Electroporation and all preceding steps were performed as described inSection A.1.1. After electroporation, the cell suspension was kept on ice for differenttimes, as indicated on the x-axis of figure A-6, before transfer of an aliquot of 0.7 mlof cell suspension to 14 ml of prewarmed, supplemented D-MEM. The viability testwas performed with an aliquot of cells removed at the times indicated; cells weremixed immediately with Trypan Blue staining solution. In order to stay within thelinear range of the CAT assay, a 2-µl aliquot of sample extract was assayed insteadof the standard 20-µl aliquot; the reaction time was reduced to 1 h. Duplicate valuesobtained during the same experiment have peen plotted in figure A-6.

Experimental Data

00

5

10

40

60

20

80

100

15

10 20 30

time after electroporation (min.)

Per

cent

14C

-CA

M c

onve

rsio

n (

)

Per

cent

Cel

l Sur

viva

l (

)

Observations:1. Transient gene expression is highest when cells are kept in the

electroporation buffer containing the transforming plasmid DNA for 1 to 2 minafter electroporation.

2. The proportion of surviving cells does not vary significantly with the time cellsare kept in the electroporation buffer.

Figure A-6. Transient Gene Expression and Cell Survival as a Function of the TimeCells are Kept in the Electroporation Buffer After Electroporation. Percent conversion of14C-CAM (O); Percent viable cells (D).

34

Experiment 7: Dependence of transient gene expression on the amount and structure ofthe transforming plasmid DNA in the presence and absence of carrier DNA (figure A-7)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pSV2CATcells . . . . . . . . . . . . . . . . . . . . . . . . . . . .COS-7 (4.2 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .HBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .500 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . .16 ms (measured with an oscilloscope)temperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

All steps were performed as described in section A.1.1, except that the differentconcentrations and forms of plasmid DNA indicated in figure A-7 were present in theelectroporation solution. In one sample, carrier DNA was omitted.Note: Less sample extract than in other experiments was assayed in order to staywithin the linear range of the assay.

Experimental Data

Panel AWith Carrier DNA

0 5 10 15 20 25 30 35 40 45 500

10

20

30

40

50

60

70

Per

cent

Panel BWithout Carrier DNA

0 5 10 15 20 25 30 35 40 45 500

10

20

30

40

50

60

70

DNA (µg/ml)

Per

cent

Figure A-7. Dependence of Transient Gene Expression on the Amount and Structure ofthe Transforming Plasmid DNA. Panel A: Experiments performed in the presence of 500 µgcarrier DNA (see Section A.1.1). A 5-µl aliquot of sample extract was assayed instead of 20 µlas stated in “Materials and Methods,” corresponding to an average of 105 cells; the reactiontime was reduced to 1 h. Panel B: Experiments performed in the absence of carrier DNA. Inthis case only 2 µl of sample extract were assayed corresponding to an average of 4 x 104

cells; the reaction time was reduced to 1 h. Supercoiled pSV2CAT (); pSV2CAT linearizedwith Sph I (O).

Observations:1. Supercoiled plasmid DNA yields higher transient gene expression than linear

DNA in the presence or in the absence of carrier DNA.2. The levels of transient gene expression are significantly higher in the

absence of carrier DNA than in the presence of carrier DNA.

35

AA.2 Electroporation of Plant Cells

Experiments were performed with tobacco and carrot protoplasts. PHBS [10 mM HEPES (pH 7.2), 150 mM NaCl, 4 mM CaCl2] containing mannitol was usedas a suspension medium during electroporation. The influence of electric fieldstrength, pulse length, DNA conformation, presence of carrier DNA and temperatureon the efficiency of transient gene expression and cell killing was studied.

A.2.1 Materials and Methods• Materials. CELLULYSIN™ was from Behring Diagnostics, La Jolla, CA;

Pectolyase Y-23 was from Seishin Pharmaceutical Co., Tokyo, Japan; andDriselase was from Plenum Scientific Co., Hackensack, NJ. NACS™ resinwas from Life Technologies, Inc. Sources of other chemicals are given in“Electroporation of Mammalian Cells, Materials.”

• Plasmid DNA. Plasmid pUC8CaMVCATAN was constructed by MichaelFromm (Stanford University); it is similar to pCaMVNEO (Fromm et al., 1985)and contains the coding region of the CAT gene under the control of thecauliflower mosaic virus 35S promoter. The plasmid was isolated from E. coliHB101 by the alkali/SDS method (Maniatis et al., 1982), further purified byNACS column chromatography to remove residual RNA (Thompson et al.,1983), and linearized by digestion with BamH I.

• Isolation of tobacco protoplasts. Protoplasts were released from leaves ofyoung tobacco plants by a 16-h digestion with 0.2% CELLULYSIN

+ 0.02% Pectolyase Y-23 [in 1 mM KCl, 1 mM CaCl2, 0.5 M mannitol, 5 mM MES (pH 5.5)] under sterile conditions. The protoplasts were purified bypassing the digest through a 70-µm nylon mesh, pelleting once (70 x g,5 min), layering the pellet over 10 ml of 18% sucrose and centrifuging.Protoplasts that accumulated at the interface were removed, washed oncewith PHBS + 0.35 M mannitol and counted with a hemacytometer. Theprotoplasts were than pelleted and resuspended in enough PHBS + 0.35 M mannitol, containing 20 µg/ml linear plasmid DNA, to give a final celldensity of 1-2 x 106 cells/ml.

• Isolation of carrot protoplasts. Cells from an exponentially growingsuspension culture of Daucus carota were digested for 5 h in a solution of 2% Driselase, 0.4 M mannitol (pH 5.0). The suspension was passed through a 70-µm nylon mesh, the protoplasts were collected by centrifugation, washedonce with PHBS + 0.25 M mannitol, resuspended in 10 ml of the samemedium, counted and pelleted again. The protoplasts were resuspended inPHBS + 0.25 M mannitol, containing 20 µg/ml linear plasmid DNA, to give afinal cell density of 1–2 x 106 cells/ml.

• Electroporation. Prior to electroporation, the cells were chilled on ice for 3 min in the electroporation chambers. Because large numbers of clean andhealthy plant protoplasts are often difficult to obtain (particularly from leaves),0.25-ml samples were used for plant cell electroporations. Electric dischargeswere delivered as single pulses and the electroporation chambers were keptat 2°C to 4°C in the chamber safe. After the discharge, electroporationchambers were kept in the chamber safe for 3 or more min or transferred toan ice bath.

• Treatment of cells after electroporation. The 0.25 ml of cell suspensionwas pipetted into tissue culture dishes, the electroporation chambers wererinsed with 0.5 ml of the appropriate electroporation buffer, and the combinedsuspension and rinse were diluted with 0.75 ml warm culture medium.Tobacco protoplasts were cultured in the dark at 27°C in K3 medium (Nagyand Maliga, 1976) containing 0.4 M glucose, at a density of 1 x 105 cells/ml.Carrot protoplasts were cultured just as the tobacco protoplasts were exceptthat the medium consisted of Eriksson’s salts containing 1 µg/ml a-naphthalene acetic acid (NAA), 0.02 µg/ml kinetin and 0.18 M sucrose.

Experimental Data

Note: It was found recently that carrotprotoplasts survive and grow better if theelectroporation chamber is rinsed withgrowth medium (supplemented Eriksson’ssalts) instead of electroporation buffer andthe cell suspension is diluted with anadditional 2.5 ml of growth medium. Thismodified protocol also markedly improvessurvival for stable transformationexperiments (data not shown).

36

• Viability tests. Evans Blue was used 48 h after electroporation to determinethe proportion of living cells.

• Preparation of cell extracts (Gorman et al., 1982). Plant cells wereharvested after 48 h by centrifugation and resuspended in 100 µl of 0.25 M Tris-HCl (pH 7.5), 0.1 mM PMSF in a 15-ml conical tube. Thesubsequent steps were the same as described in section A.1.2.

• Assay for Chloramphenicol Acetyl Transferase (CAT) (Gorman et al.,1982). The assay was performed exactly as described in section A.1.2,Assay for Chloramphenicol Acetyl Transferase, except that a 50-µl aliquot ofcell extract was used.As in the experiments with mammalian cells, the CAT activity is expressed asa percent of 14C-CAM converted to acetylated forms using the formula

% conversion of 14C-CAM = cpm in acetylated spots x 100/total cpm

Note: CAT activities above 60% conversion of 14C-CAM are beyond the linearrange of the assay and therefore are underestimates of the true activity.

Experimental Data

37

AA.2.2 Results and Observations

Experiment 8: Transient gene expression and cell survival as a function of field strengthand pulse length for tobacco leaf cell protoplasts (figure A-8)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . .pUC8CaMVCATAN (linear)cells . . . . . . . . . . . . . . . .tobacco leaf protoplasts (1 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PHBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250 to 750 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100 to 300 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to 112 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Experimental Data

00

20

40

60

80

100

20 40 60 80 100 120

Pulse Length (ms)

Per

cent

Figure A-8. Transient Gene Expression in Tobacco Leaf Cell Protoplasts and CellSurvival as a Function of Field Strength and Pulse Length. Percent conversion of 14C-CAM at 250 (), 500 () and 750 V/cm (), respectively; Percent Viable cells at 250 (D),500 (D) and 750 V/cm (D), respectively.

Observations:1. Transient gene expression is highly dependent on field strength and pulse

length. At 250 V/cm transient CAT expression increases consistently withincreasing pulse length. At 500 V/cm, the peak in CAT expression isobserved at a pulse length of about 10 ms, and at 750 V/cm, the peaktransient gene expression is observed at a pulse length of approximately 2.5 ms.

2. The percentage of viable cells decreases steadily with increasing pulselength at all field strengths tested. Cell killing increases with higher fieldstrengths. Significant levels of transient gene expression are observed only ifa portion of the cells are killed as a consequence of the electric pulse.

38

Experiment 9: Transient gene expression and cell survival as a function of pulse lengthfor carrot cell protoplasts (figure A-9)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . .pUC8CaMVCATAN (linear)cells . . . . . . . . . . . . . . . . . . . . .carrot protoplasts (2 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PHBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .750 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .300 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .0 to 112 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

Experimental Data

00

20

40

60

80

100

20 40 60 80 100 120

Pulse Length (ms)

Per

cent

Figure A-9. Transient Gene Expression in Carrot Cell Protoplasts and Cell Survival as aFunction of Pulse Length. Percent conversion of 14C-CAM (O); Percent Viable cells (D).

Observation:Transient gene expression increases with increasing pulse length and thepercentage of viable cells decreases steadily with increasing pulse length.

39

Experiment 10: Effect of DNA form and concentration on transient gene expression incarrot cell protoplasts (table A-2)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pUC8CaMVCATANcells . . . . . . . . . . . . . . . . . . . . .carrot protoplasts (2 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PHBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .750 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .300 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 mstemperature at electroporation . . . . . . . . . . . . .0 to 4°C (ice water)

The experiment was carried out as described in section A.2.1. DNA was linearizedby digestion with BamH I. Carrier DNA (50 µg/ml heat-denatured salmon spermDNA) was added to the sample just before electroporation. Control samples werecells resuspended in PHBS in the presence of linearized plasmid but notelectroporated. The values given in table A-2 are the average of duplicates obtainedin the same experiment.

Table A-2. Effect of DNA Form and Concentration on CAT Expression in Carrot CellProtoplasts After Electroporation in the Presence or Absence of Carrier DNA.

CAT

DNA Activity

Concentration Carrier (% Conversion of

DNA Form (µg/ml) Electroporation DNA 14C-CAM)

Linear 20 – – 0.4

Linear 20 + – 2.0 6 0.3

Linear 40 + – 2.7 6 0.5

Linear 20 + + 3.6 6 0.5

Linear 20 – – 0.3 6 0.1

Linear 20 + – 2.2 6 0.1

Supercoiled 20 + – 4.4 6 1.4

Observations:1. Doubling the concentration of the linearized plasmid DNA from 20 to 40 µg/ml

increased CAT expression by about 40%.2. The addition of carrier DNA increased expression by approximately 80%.3. Supercoiled DNA yields consistently better expression than the linearized

form of the plasmid; in the experiment shown here expression is doubled.

AExperimental Data

40

Experiment 11: Effect of temperature on transient gene expression in carrot cellprotoplasts (table A-3)

Experimental conditions:plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .pUC8CaMVCATANcells . . . . . . . . . . . . . . . . . . . . .carrot protoplasts (2 x 106 cells/ml)electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .PHBSfield strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .750 V/cmvoltage setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .300 VV setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .LOW Vpulse length . . . . . . . . . . . . . . . .28 ms (at 0°C) or 14 ms (at 20°C)temperature at electroporation . . . . . . .0 to 4°C (ice water) or 20°C

The experiment was carried out as described in section A.2.1, with the followingexception:

1. The initial equilibrium period of cells in electroporation buffer containing DNAwas 5 min rather than 3 min.

2. Two different temperatures were tested. For the experiment at 0 to 4°C, theelectroporation chambers were kept in ice water; for the experiment at 20°C,the electroporation chambers were kept in the chamber safe surrounded byair of ambient temperature.

The values given in Table A-3 are derived from triplicate samples in the sameexperiment.

Table A-3. Effect of Temperature on CAT Expression in Carrot Cell Protoplasts

Temperature CAT Activity Viable Cells(°C) (% Conversion of 14C-CAM) (%)

0–4 3.2 60.4 37 6 2

20 20.0 6 3.6 38 6 2

Control values of 0.4% conversion of 14C-CAM were obtained at 0°C and 20°C,respectively. Control samples were not electroporated.

Observation:Transient gene expression was significantly better when electroporation was carriedout at room temperature rather than at 0°C to 4°C.

Experimental Data

41

A.3 Discussion

Electroporation is a simple and reliable method for stimulating the uptake of DNA bymammalian and plant cells. The levels of transient gene expression and stabletransformation achieved with this method are equal or better than those achieved byconventional methods such as calcium phosphate/DNA coprecipitation (Shimizuand Watanabe, 1986; Chu et al., 1987). Electroporation is also effective instimulating transfection with cells that do not respond to conventional methods(Potter et al., 1984; Reiss et al., 1986; Toneguzzo et al., 1986; Tur-Kaspa et al.,1986). Another advantage of electroporation is the ability to make cells permeableto RNA (Okada et al., 1986), nucleotides (Sokoloski et al., 1986), and proteins anddyes (Sowers and Lieber, 1986).

We have investigated several key parameters for electroporation-facilitatedtransfection as measured by the expression of CAT activity 48 and 70 h afterelectroporation of mammalian and plant cells, respectively. Although conditionsmust be optimized for each cell type, several general guidelines can be deducedfrom the results presented in the experiments described here and the resultspublished by other laboratories. The findings appear to be relevant for both transientgene expression and stable integration of foreign DNA (Toneguzzo et al., 1986; Chuet al., 1987). The results obtained with mammalian and plant cells are discussed inthe following sections.

A.3.1 Mammalian Cells

In experiments 1 to 7, we have investigated what seem to be the most importantvariables for the transfer of DNA into mammalian cells via electroporation. We haveattempted to carry out a coherent series of experiments, varying one parameter at atime. Although in all of the experiments described here COS-7 cells and pSV2CATDNA have been used, similar results have been obtained with mouse L cells andpRSVCAT DNA (data not shown).

• Field strength and pulse length. The efficiency of DNA uptake has beenreported to be a function of the applied field strength as well as the pulselength (Zimmermann, 1982; Potter et al., 1984). One important observationfrom experiments 1, 2, 3 and 5 is that a high level of transient geneexpression is associated with some pulse-induced cell killing. However, it isclear from the data that the absolute values vary. It appears that this isprimarily due to variations in cell growth and the treatment of cells before andafter electroporation. Another important observation is that shorter pulses canpartially compensate for higher field strengths and lower field strengths canpartially compensate for longer pulses. However, both field strength and pulselength must be optimized for maximal CAT expression (experiments 1 and 2). Considerable differences in optimal conditions have been observedfor different cell types (data not shown) and even different cell preparations(see, for example, experiments 1 and 2).

• Ionic strength and composition of the medium. The composition of themedium in which electroporation is performed is critical. A comparison of alow ionic strength buffer (HBM) with a high ionic strength buffer (HBS) hasbeen performed. The results of experiment 3 indicate that electroporation canbe carried out successfully in high ionic strength buffers, at relatively low fieldstrengths, and that such buffers actually yield higher transient geneexpression than their low ionic strength counterparts. Moreover, experiment 4has shown that electroporation is even more efficient in D-MEM; thus, theneed for a special electroporation buffer is eliminated.

• Temperature. The temperature before, during and after electroporation has amarked effect on transient gene expression. CAT expression is considerablyhigher (particularly at shorter pulse lengths) when mammalian cells are kept atroom temperature rather than chil led in ice water (experiment 5).

AExperimental Data

42

Electroporation at temperatures other than about 2°C and 22°C has not beeninvestigated; it is possible that other temperatures are optimal. There isevidence that the temperature dependence might vary strongly with the celltype. Potter et al. (1984) reported higher efficiency of stable transformation ofM12 cells (spontaneous mouse B-cell lymphoma) when electroporation wascarried out at about 2°C rather than at room temperature. The authorsspeculated that this might be due to reduced membrane fluidity at lowtemperatures, resulting in a longer half-life of open “pores” through which DNAcould enter into the cell. Although the validity of this hypothesis needs to beestablished, the apparent contradiction between the results of Potter et al. andthe results reported here points to the fact that transient gene expression is avery indirect measure of DNA uptake. No conclusions can be drawn from suchexperiments about the efficiency and the mechanism of DNA transfer intocells. Chu et al. (1987) have also obtained higher levels of transient geneexpression for several mammalian cell lines when electroporation wasperformed at 20°C (compared with electroporation at 0°C).

• DNA concentration and conformation. Supercoiled pSV2CAT DNA yieldshigher levels of transient gene expression than the same plasmid cut with Sph I (experiment 7). This observation contradicts a number of findingsreported in the literature which indicate that linear DNAs yield higher levels oftransient gene expression and stable transformation than supercoiled DNAs(Neumann et al., 1982; Potter et al., 1984; Toneguzzo et al., 1986). We havealso observed higher transient gene expression in carrot cell protoplasts inresponse to transfection with supercoiled DNA rather than linear DNA(experiment 10). At this time we do not have an explanation for thesediscrepancies.

Levels of transient gene expression are higher in the absence of carrier DNAthan in the presence of carrier DNA (figures 7A and 7B). However, the purityof the plasmid DNA may markedly affect the response to the addition ofcarrier DNA. The plasmid DNA purified as described in “Materials andMethods” contains a significant amount of RNA and possibly othercontaminants. Plasmid DNA purified further than that used in this experimentdid not display the reduction in transient gene expression upon addition ofcarrier DNA (data not shown).

• Post-electroporation incubation time. Using HBS with 10 µg/ml plasmidDNA, maximal CAT activity is observed when cells are held in this solution forabout 2 min after electroporation (experiment 6). In most publishedprocedures, cells have been held on ice for 3 to 10 min after electroporationbefore dilution into warm medium (Fromm et al., 1985; Smithies et al., 1985;Reiss et al., 1986; Riggs and Bates, 1986; Tur-Kaspa et al., 1986). From theresults reported here, the period of time between electroporation and thereturn of cells to the growth conditions is an important parameter which shouldbe taken into account when optimizing electroporation-facilitated DNAtransfer.

A.3.2 Plant CellsIn designing and performing electroporation experiments with plant cells, the samerationale was used as for mammalian cells. Protoplasts prepared from tobaccoleaves or from carrot cells grown in suspension culture were used.

• Field strength and pulse length (experiments 8 and 9). The basic effects ofdifferent field strengths and pulse lengths for plant cells are the same asobserved for COS-7 cells: increasing field strength and pulse length causesdeath of an increasing portion of the cell population; maximum CAT activity isobserved when a significant portion of the cells are killed as a consequence ofthe electric pulse; and, within certain limits, higher field strengths cancompensate for shorter pulses and longer pulses can compensate for lowerfield strengths.

Experimental Data

Note: DNA was further purified by NACScolumn chromatography which eliminatedessential ly all contaminating RNA(Thompson et al., 1983).

43

Good quality plant cell protoplasts are more difficult to prepare than goodquality mammalian cells. Experiments performed with marginal or poor qualityprotoplasts resulted in massive cell death but little or no CAT geneexpression. Optimal field strength and pulse length vary for cells of differentorigins. Maximal CAT activity in tobacco leaf protoplasts at 500 and 750 V/cmis obtained with short pulses (experiment 8). For carrot protoplasts, maximalCAT activity at 750 V/cm is obtained with much longer pulses (experiment 9).The absolute level of CAT activity also varies for the two different cell species.The causes for these differences require further investigation.

• Temperature. The temperature of the electroporation medium before, duringand after electroporation has a significant effect on transient gene expressionof plant cells. When the capacitance setting is held constant (at 800 µF),carrot protoplasts yield six-fold more CAT activity at 20°C than at 0°C to 4°C(experiment 11). Similar results have been obtained with tobacco leafprotoplasts (data not shown). This effect does not appear to be the result ofdifferential cell killing by pulses delivered at the different temperatures. Thus,it probably reflects an increase in DNA uptake at higher temperatures.

• DNA concentration and conformation; effect of carrier DNA. Doubling theconcentration of linear DNA from 20 to 40 µg/ml resulted in an increase ofCAT activity of about 40%, indicating that even higher DNA concentrationsmight not yield proportionately higher CAT activity. Fromm et al. (1985) haveshown that both transient gene expression and stable transformation increasein parallel with increasing DNA concentrations. The addition of carrier DNA toa sample containing 20 µg/ml of plasmid DNA enhanced CAT activity byalmost 80%. Supercoiled DNA yielded consistently higher CAT activity thanthe linearized form of the plasmid. To the best of our knowledge, systematicstudies have not been published on the influence of DNA conformation onplant cell transformation. However, both linear DNA (Riggs and Bates, 1986)and supercoiled DNA (Fromm et al., 1985 and 1986) have been usedsuccessfully in the range of 10 to 50 µg/ml.

A.3.3 Composition of the electroporation buffer

Much of the early work on electroporation was performed with low conductivitybuffers containing low concentration of salts and a nonmetabolized carbohydrate,such as mannitol, to make the solution isotonic (Zimmermann, 1982). Successfulelectroporation and DNA uptake in mannitol solutions has been reported for plantcells (Riggs and Bates, 1986). However, as in the case of mammalian cells,buffered salt solutions have yielded higher levels of transient gene expression(Fromm et al., 1985 and 1986; experiments 8 and 9). Using the standardexperimental protocol described here, some CAT activity (0.6% to 1.1% conversionof 14C-CAM per 10 cells) was obtained with carrot protoplasts in 0.5 M mannitolfollowing a 750 V/cm, ~30 ms pulse. However, much greater expression (16% to 60% conversion) resulted when electroporation was carried out in PHBS atthe same field strength and pulse length.

AExperimental Data

44

A.4 References1. Chu, G., Hayakawa, H. and Berg, P. (1987). Nucleic Acids Res. 15, 1311.2. Fromm, M., Taylor, L.P. and Walbot, V. (1985) Proc. Natl. Acad. Sci. USA 82,

5824.3. Fromm, M., Taylor, L.P. and Walbot, V. (1986) Nature 319, 791.4. Gorman, C.M., Moffat, L.F. and Howard, B.H. (1982) Mol. Cell. Biol. 2, 1044.5. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning:

A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.6. Nagy, J.I. and Maliga, P. (1976) Zeit. Pflanzenphysiol. 78, 453.7. Neumann, E., Schaefer-Ridder, M., Wang, Y. and Hofschneider, P.H. (1982)

EMBO J. 1, 841.8. Okada, K., Nagata, T. and Takebe, I. (1986) Plant Cell Physiol. 27, 619.9. Potter, H., Weir, L. and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161.10. Reiss, M., Jastreboff, M.M., Bertino, J.R. and Narayanan, R. (1986) Biochem.

Biophys. Res. Commun. 137, 244.11. Riggs, C.D. and Bates, G.W. (1986), Proc. Natl. Acad. Sci. USA 83, 5602.12. Shimizu, H. and Watanabe, T. (1986) Medical Immunology 11, 105.13. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. and Kucherlapati,

R.S. (1985) Nature 317, 230.14. Sokoloski, J.A., Jastreeboff, M.M., Bertino, J.R., Sartorelli, A.C. and Narayanan,

R. (1986) Anal. Biochem. 158, 272.15. Sowers, A.E. and Liever, M.R. (1986) FEBS Lett. 205, 179.16. Thompson, J.A., Blakesley, R.W., Doran, K., Hough, C.J. and Wells, R.D.

(1983). In: Methods in Enzymology: Recombinant DNA, Part B. R. Wu, L. Grossman and K. Moldave (eds.), Volume 100, 6-38, Academic Press, NewYork.

17. Toneguzzo, F., Hayday, A.C. and Keating, A. (1986) Mol. Cell. Biol. 6, 703.18. Tur-Kaspa, R., Teicher, L., Levine, B.J., Skoultchi, A.I. and Shafritz, D.A. (1986)

Mol. Cell. Biol. 6, 716.19. Zimmerman, U. (1982) Biochem. Biophys. Acta 694, 227.

Experimental Data

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