Gene transfer into mouse lyoma cells by electroporation in high electric fields

Gene transfer into

mouse lyoma

cells by electroporation in high electric fields

E.Neumann*, M.Schaefer-Ridder, Y.Wang, and P.H.Hofschneider

Max-Planck-Institut ftirBiochemnie,D-8033Martinsried/Muinchen, FRG Communicated by E.Neumann

Received on 30 June 1982

Electric impulses (8 kV/cm, 5

tis)

were found to increase greatly theuptake of DNA intocells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK)gene is added to a suspension of mouse L cells deficient intheTKgene and thecells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium.At20°C after theapplicationof threesuc- cessiveelectricimpulses followed by 10 min to allow DNA en- try thereresult 95 ( ^-3) transfonnants per106 cells andper 1.2ytg DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple,easilyap- plicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells inhigh electric fields is proposed. According to this 'electro- poration model' the interaction of the extemal electric field with the lipid dipoles of a pore configuration induces and stabilizesthepermeation sites and thus enhances cross mem- branetransport.

Key words: electric impulses/gene transfer/mouse L cells/

pBR322plasmid

Introduction

A variety of biochemical methods have been developed to transfer genes into eukaryotic cells including incubation of the recipientcells with co-precipitates of DNA and Ca-phos- phate (Graham and van der Eb, 1973), direct injection of genes into the nucleus of the recipient cell (Diacumakos,

1973),useof viralvectors(Hamerand Leder, 1979; Mulligan etal., 1979), andapplicationof liposomesasvehicles for gene transfer (Fraley et al., 1980; Wong et al., 1980; Schafer- Ridderetal., 1982).

Here wereport a physical method that leads to anenor- mous enhancement ofDNA transport acrosscellular mem- branes. Thenew techniquestems from the observation that biomembranes are made transiently morepermeable by the action of short electricimpulsesaboveacertain fieldstrength, without damaging the membrane structures (Neumann and Rosenheck, 1972, 1973; Zimmermannet al., 1973; Kinosita and Tsong, 1977). The electrically induced increase in permeability leadsto a transient exchange ofmatter across theperturbed membrane structures. Theelectric field effect ontransport is clearly a membrane phenomenon (Sale and Hamilton, 1968; Rosenheck et al., 1975; Lindner et al., 1977). Electricimpulsesnotonlyinduceorenhanceexchange ofmaterialacrossmembranesbuttheyalsocausemembrane- membrane fusion whentwomembranesarein close contact

with each other (Neumann etal., 1980; Scheurichand Zim- mermann, 1980;Weberetal., 1981).

*To whomreprintrequestsshouldbesent.

Whencircular or linear DNA carryingthe thymidine kinase (TK) geneis added to a culture of mutant mousecells(LTK-

cells) deficient in this gene, which is then subjected to a se- quenceofelectric field pulses at 20°C the LTK- cells take up large amountsof the plasmid DNA within a period of ^- 10 min after pulsing. The newly acquired TK activity is demonstrated by the survival of the transformed cells in a selection medium (Pellicer etaL, 1978). The firstelectric field experiments with LTK- cells and DNA containing the TK gene showed that there is enhanced colony formation after pulsing (Wong and Neumann, 1982). However, the number of stable transformants in these first experiments was rather low.

Here, data are presented which lead to the suggestion of some optimum conditions for electrically mediated gene transfer. They show that theelectric impulse method is a sim- ple, easily applicableand very efficient technique to transfer linear and circular DNA into recipient cells. The electric method to increase membrane permeability for artificial DNAtransfer should be generallyapplicable, and particularly valuable with those celllines with which biochemical methods have notbeen successful.

Results

The electrically mediated DNA transfer into LTK- cells stronglydepends on the initialfield strength of the electric im- pulses(Materials andmethods). AsshowninFigure1,there is a threshold of -6-7 kV/cm and an optimum field strength rangeof 8 (^- 0.5) kV/cm for DNA transfer leading tocolony formation intheHATselection medium. At higher field strengthsthe cells areirreversiblydamaged (Wong and Neumann, 1982; seealsoSale and Hamilton, 1968).

Although the presence of divalent ions likeMg2+ increases theamount of DNA bound tothe cell surfaces (Wong and Neumann, 1982), Mg2+ actually reduces and finally prevents gene transfer. Figure 2shows that increasing concentrations ofMgCl2decrease the number ofcolonies; at30mMMgCl2 the pulsed and unpulsed samples give the same number of colonies: 3 ( 3) per 106cellsand per 1.2

,g

DNA.When no

6 4

2 /

0 2 4 6 8 10

Eo JkV cm-1

Fig. 1.Thedifference(,AQ)inthenumber ofcolonies(pulsedminusun-

pulsed sample)per 3.8x 106pulsedcellsper 1AgDNAat20 mMMgCl2in HBS,asafunctionoftheinitialfieldintensityEoofexponentiallydecay- ingimpulseswith rE = 5 As;seeequation (13)ofthetext.

841

E.Neumann et al.

MgCl2 isadded tothe mixture of cells andDNAtheyield is 95 (-i3)colonies per 106 cells and per 1.2

;tg

^DNA.

Toexploreoptimum conditions for thiselectrically induced genetransfer, non-optimum conditionswithrespect toMgCl2 were chosen; eventual increases in the colony yield with in- creasingamounts of added DNAand higher cell densitities should be easily recognizable at 20 mM MgCl2 (Figure 2).

Figure 3 shows that thecolony density increases with increas- ing amounts of DNA,both for linear and circular DNA. The numbers of colonies obtained with linear DNA are higher than those with circular DNA, for both the pulsed and the unpulsed samples.

At zero MgCl2 theyield from amixture of 20

jig

circular DNAand 4x 105cells/0.4mlis 30 ( 3)colonies per 0.1 ml of the(pulsed) suspension. Since only 0.29mlof the 0.4ml sample volume are actually pulsed, this result means 500 (X20) transformants/106 cells/5

Atg

DNA. With the linear DNA the estimated yield is - 103 transformants/106 cells/5 yg DNA.

Figure4 shows that the number ofcolonies alsodepends

100

Q

50

CMgC12,

mM

Fig.2.Numberof colonies(Q)per106 pulsed cellsand per 1.2jgDNAin HBS,asafunction oftheMgCl2 concentration; *,pulsed; 0,unpulsed (control).

a.

on the time interval between the electric pulses and the transfer of the pulsed cells to Dulbecco's minimal essential medium (DMEM; Materials and methods). At 20°C the saturationvalue is reached after -10min. This observation is inline with thefact that electrically induced perturbations of vesicle and cell membranesarelong-lived compared with the duration of the impulse (Rosenheck etal., 1975; Zimmer- mann etal., 1976; Lindneretal., 1977).

Discussion Biological aspects

The resultsof thisstudyon DNA transfer into LTK - cells suggestsomegeneralguidelines for the determination ofop- timum conditions forelectrically mediated gene transfer. In the case of LTK-mousecells, Mg2+ionsmustbe absent (see also Grahamand van derEb, 1973). At least threepulsesof aninitial fieldintensity of 8(+5)kV/cm and apulse decay time of ^- 5

As

should beappliedat20°C. Thepulsedsample shouldbetransferredintoselection mediumonly10minafter the pulsing. Asexpected, cell density andDNAconcentration should beashighaspossible; e.g. 5 x 107cells/ml and 50

jig

DNA/ml(cf. Figure3).

Simple incubation of LTK- cells withDNA occasionally leads to the formation of a few transformants (Figure 3).

Therefore, DNA adsorbed on the cell surface is able to penetrate intothe cellinterior;theyieldofcolonies,however, is low compared withthe colony yieldafterelectricpulsing.

The detailed optimum conditions for the electric gene transfer into cells willdepend onthe cell type and the sizeof the cells (seebelow)andmustbeexploredforeachcasealong the linesdescribed above. Even fornon-optimumconditions theyield obtained with thefieldtechniquecompareswell with that ofthe presentbiochemical methods. The mainadvantage ofthe electric fieldprocedureis,however,itseaseofapplica- tion and as a physical method it should be generally ap- plicable.

Biophysicalaspects

The mechanism ofcross membrane transport ofDNA is eitheranunspecificmembrane processorit maybespecifical- lymediated by permeases that areactivated in high electric fields. A biological membrane is a co-operatively stabilized organizationoflipids and proteinswhich containsdynamic, locally limited structural 'defects'. These local disorders are the candidates for the onset of further, electric-field induced perturbations, leading to permeation sites for enhanced materialexchange. The additional-electric potential difference

-

[DNA],

pgml-

Fig.3.Numberof colonies(Lo)as a function ofthe DNA concentration in themixture of5x107cells/ml as afunctionofthe DNA concentration, 20mMMgCl2,HBS. (a)circular DNA, *; unpulsed control, L; (b) linear DNA, A;unpulsed control,A.

842

iu u -

5

^-

0

0

0

5 10 15

t, min

Fig.4.Thedifference(AQ) in the number ofcolonies (pulsed minus un- pulsed)asafunctionof the incubation time after the pulsing.Experimental conditions:5x 106cells/ml, 12Ag^{DNA/ml,20 mM}MgCl2,HBS,200C.

A V which develops across the membrane isgivenby:

AV = AV(O) cos0 (1)

Therefore, the largest valuesof the field force vectorexist at the 'pole caps' where0 = 0; see Figure 5.

The maximumpotential difference, AVmax,whicha mem- brane of acell ororganelle experiences, depends on the cell size and isapproximately givenby:

AVmax = 3 Eo r (2)

2

whereEois the initial field strength andris theradius of the vesicle (Fricke 1953).When high electricfields are applied for only a shorttime ( < 5

As)

the electric impulse lead to arever- sible 'pore formation', probablyin thelipid partof the mem- branes (Benz and Zimmermann, 1981; Teissie and Tsong, 1981) and various models have been proposed (Crowley, 1973; Zimmermann et al., 1981;Abidor et al., 1979).

The presence of anexternal electric field will favour charge anddipole configurations that leadstolarger dipolemoment components inthe field direction. This inturnmayleadtoa thinning of membranepatches and finally to a hole, as pro- posed inFigure 5. The structural changes in a membrane do- main forming a hole can be subjected to a general thermo- dynamic analysis. To a first approximation we may use a two-statemodel forthe 'electroporation' process:

[closed structure]

,- [open

structurel (3)

k(c)

The stability constant for this two-state model is given by the distribution of all membrane components Bj in the closed configuration,

Bj(C),

and in the openconfiguration,

Bj(o),

^ac-

cordingto:

K = Hl

[Bj(o)]/rI[Bj(C)]

(4)

Forastable, not permeable, membranedomain the inequality

(b)

(c)

(d) (e)

Fig. 5. Diagram of the electrically induced transfer ofDNAintocellsby theelectroporationprocess.(a) Model ofasphericalbiologicalcell with nucleusandanexternal DNAonthecellmembranesurface,relativetothe direction ofhomogeneouselectric fieldE;0is theapical anglewithin which the membranestructureexperiencesanabove-threshold fieldintensityand becomespermeable (cap). (b) Permeation of theDNAthrough^apathway inducedbyelectroporation. (c)DNAassociation with the nucleus. (d) Unperturbed lipiddomain inamembrane; (e)hole(pore),stabilizedbya

favourablelipidheadgroupdipoleinteraction with the electric field.

K < 1 holds, i.e., the concentration of hole areas is very small.

When wedenote the stabilityconstant of a single compo- nentinteraction (of the B.) by sj andintroduce an overall co- operativity parameter a, tien equation (4) may be rewrittenin terms of theseZimm-Bragg parameters as:

K =

ausj

(5)

The netstability constant for the permeable structureis thus the product over all molecular contact sitesj.Thenucleation factor a, with a < 1 representing the low probability of nucleating a hole, assures a very steep structural transition curve, as may be derived from the data in Figure 1.

As anequilibrium constant, Kwilldependontheintensive statevariables, temperatures (7), pressure (P), and apossibly applied electric field (E). The dependence of the domain stabilityonthese state parameters may be generally writtenin adifferential form as:

dlIn

K=lTI

dT+

a!!

dP+

E\dE

⁽⁶⁾

O P,E T,E ' 'P,T

Introducing the general van't Hoff relations, equation (6) is rewritten as:

d

ln

K dT- dP +

AMdE

(7)

R12 RT RT

Inthisrelationship the overall reaction enthalpy AH= EjAHj is the sum of the component contributions AHj associated with sj; A V= E AV isthe overall volume change, and AM= 2i-AM,is theoverallreactiondipole moment representing the difference between the dipole moments of the reaction pro- ducts (hole configuration) and the components of the closed structure. Fromequation(7) itisclearlyseenthat a structural distribution, represented by K, may be changed by a change ofthe temperature, or ofthe pressure, or.bythe application of anexternal electric field, provided that the overall reaction quantitiesAH, AV, and AM have finite values, respectively.

Denoting by0the degree ofstructural transition, then the overall equilibrium constant of the two-state model in equa- tion (3) is generally given by

K= 0/(l -0) (8)

Notethat0 =

E[Bj(0)I/(F[B-(o)]

+

E[Bj(c)]).

It is now readily shown that the change in the degree of transformation is given by (Neumann,_1982):

d0= 0(1 -0) tAH dT- dP +

AMdEI

(9)

R 7 RT RT

This relationship demonstrates that the extent of a structural changedepends, in addition, onthe 'position' value0of the distribution. Clearly, a maximum relative change is produced if0 < <1, i.e., ifthe entire membraneinitiallyis in a closed configuration.

Equation(9)rationalizes the structural domain transitions thermodynamically, but also the enormous difference in the kinetics of pore formation and annealing can be generally formulated. The overall rate constants for the forward and backward direction in equation (3) may be denoted by k(o) and

k(c)q

respectively. Werecall that the reaction dipolemo- ment AM = (aM/3O) is the difference between the dipole contributions of theproductsandthe reactions.Forequation (3), wehave:

AM ⁼ O (10)

E.Neumann et al.

where M(o) is the macroscopic dipole moment of the open configuration, M(c) that of the closed structure, and t is DeDonder's general reaction variable describing theextent of structural transition. Since the open structure is favored in the field, the inequality:

(aiO

)E>Ca)

⁽¹¹⁾

musthold for the field strength range E 2 Eth,where Eth is thethreshold value.

The formation ofcolonies ofLTK + cells in the selection medium is certainly the result of DNA penetration through permeation sites induced by the electric field actiononthecell membranes. The steep dependence of thecolony density, Qc,

on the field strength, as shown in Figure 1, reflects a steep dependence of 0 onE, and thus alargevalue of AMwhich, mostlikely, is caused byaco-operative transitioninvolvinga larger number of membrane constituents in a (co-operative) domain.

The structural change leading to an open holeconfigura- tion is rapidly introduced by the field within a few micro- seconds. The annealing of the structural perturbation after the electric field has decayed tozero, is associated with time constants inthe second and minute range. Obviouslythere is agreatrate ofenhancement for a transition of thetypegiven in equation (3) in the presence of the field. Now, straight- forward thermodynamics describes the field dependence of the opening rate constantaccording to(Neumann, 1982):

k(o)(E) = k(o) (0) * exp[(R7")

1ff(aM(0)/at)AdE]

(12) Clearlyat E = 0, the low background DNA penetration of the unpulsed sample must mean that k(o) (0) << k(c) (0) holds. The annealing process after thepulsing (whereE_= 0) issimply described by k(c) (0).As seen inequation (12),k(o)is greatly enhanced in the field when M(o) increasesduring the structural transition.

Theexpressions (9)- (12) are fundamental relations for a thermodynamic and kinetic analysis ofelectric field-induced changes in the structure ofmembranes ingeneral; they may be specifically applied to describe particle transfer through cellular membranes such as the electrically mediated DNA transfer intocells.

Materialsand methods

CellsandDNA

MouseLTK-cellsweregrown in DMEMwithlO0o fetal calf serum. The cells were trypsinized, washed,andresuspended in HEPESbuffered saline (HBS: 140mMNaCl,25mMHEPES, 0.75mMNa2HPO4,pH7.1at20°C) toafinal celldensityof 5x107cells/ml.

The recombinantplasmid,pAGO,carryinganactive TK gene (2 kb of the PvuII fragment ofHSV I) inserted in thePvuII site ofpBR322 (Colbere- Garapinetal., 1979)wasused fortransformationexperiments, either in its supercoiledformorinthe linear form bydigestion withHindIII.

Thesolutions of theLTK- cells were incubated for 10 min with various amountsofDNA, upto20AgDNA/0.4ml, with and withoutMgCl2,before transferring the solutiontothe electric measuring cell for pulsing. To maintain sterileconditions thesamplecell was washed with70% alcohol; gloves and mouthprotection are necessary to avoid contamination.

About 10 min after the electric impulses, aliquots of 100jilof the cell suspensionweretransferred into 10 ml DMEM and seeded into two 10-cm Petridishes. To select for stable transformants the cell monolayers were in- cubated after 24 h with HAT medium (hypoxanthine, aminopterin, thymidine)under standard conditions (Pellicer et al., 1978). After 2 weeks in HATselection medium the TK-positive colonies were counted.

Theelectric impulsemethod

Themeasuringcell ofanelectricdischargecircuit(Figure 6)is filled with 0.35 ml DNA cell suspension and then subjected to a series ofimpulses.

0 EE t 0 t

Fig.6.Diagramof themeasuringcell(MC), filling volume0.35ml, forthe electricimpulse experiments; dischargecircuit forcapacitor discharge (C),

oralternatively,cabledischarge (Cb); G, high voltagegenerator;SP, spark

gap.Timecoursesofthe electric field forcapacitor discharge and forrec-

tangular pulsesfrom thecabledischarge, respectively.

Usuallythreesuccessivepulsesareappliedataninterval of 3s.Thespecialcell constructionpermits therequiredbubble-freeclosureof themeasuring cell;

0.29 ml of the solution isexposedtothe electric field.

Ahigh voltage generatorprovides theelectricalenergy, which at first is stored inahigh voltage capacitoror,alternatively,ina1000mcoaxialcable.

The discharge is triggered manually by a spark-gap (Figure 6). The temperatureofthe thermostatedsamplecell is20°C.

Incaseof thecapacitor discharge,theinductance of thecircuit issolow that thevoltage, U,acrosstheconductingsolutionbetween theelectrodes of thesamplecelldecays exponentiallyfromaninitialvalueU0.Since the electric fieldstrengthis definedbyE= U/d,where d is the distancebetween the elec- trodes,theexponential decayof the field forceisgiven by:

E(t)= Eoexp(^-t/TE) (13)

Thefield-decaytime constantisTE =RC;where C is thecapacitanceof the discharge capacitor (C= 12, 21,or52nF),Eo= Uo/dand R is theresistance ofthedischargeunit. The resistance of thesamplecell(>200 9) usuallydeter- mines the circuit resistance.

Because of the finite conductance of thesolutions the electricenergyofthe impulse dissipates.The finaltemperature increase isgiven by:

AT= 0.5 C

U2/(pcpv)

(14)

whereQis thedensityof thesolution,cpis the heatcapacity,andv=0.29 cm3 is the effectivevolume thatexperiencesthe electricfield. Herewemayusethe approximations Q 1 g/cm,cp = 4.12 J/(gK) (see, e.g., Neumann and Rosenheck, 1972).

As shown above(Results), optimumconditions forsuccessfulgenetransfer intoLTK-cellsare U0 = 8kV, C = 21nF,and R ⁼ 250Q. Thus, with equation (13),thefield-decayconstantis TE= 5.4its. Fromequation (14)we obtain AT= 0.55°C;thusthetemperature increaseperpulseisnegligible.

Acknowledgements

The technical help of U.Santarius, I.Dick, and H.Goehde is gratefully acknowledged.Wethank H.Riesemann forthe isolation oftheplasmidDNA.

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