An improved electrotransfection method using square shaped electric impulsions

Vol. 151, No. 3,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 982-990

March 30,1988

AN IMPROVED ELECTROTRANSFECTION METHOD USING SQUARE SHAPED ELECTRIC IMPULSIONS

Carmen

Francoise Presse*, Anne Quillet*, Lluis Mir**, Marchiol-Fournigault*, Jean Feunteun** and Didier Fradelizi* * Laboratoire d'Immunologie CNRS UAII56/IGR/INSERM Institut Gustave Roussy, 94805 VILLEJUIF

** Laboratoire

d'Oncologie Moleculaire CNRS UAII58/IGR/INSERM Institut Gustave Roussy, 94805 VILLEJUIF

Received January 25, 1988 Transfection of DNA into non adherent cells can be achieved by electropermeation. Previously published results, partially successful, were obtained using exponential decaying electric impulsions. However, one limitation of this technique has been the damaging effect of t h i s type of impulsions resulting in Door cell recovery. We report hereby the electropermeation of human lymphoblastoid cell lines using a commercially available electropulsator delivering repeated, short, high voltage, square shaped, electric pulses. The parameters of transfection have been optimized using the "Lucifer Yellow Permeation Assay". With the optimum electric parameters, virtually all the cells were permeated and at least 70 % survived the shocking conditions. Both transient expression and permanent integration and expression of DNA was observed. © 1988AcademicPress,Inc.

Classical transfection DEAE dextran

(2,3)

poorly efficient expression of

and protoplasm fusion methods (4,5), have proved

for

the

genetic

transfection results method (6)

using

techniques including calcium phosphate (i),

obtention

material

of

either transient or stable

into non adherent cells .Successful

have been reported with the electrotransfection

carefully

defined

parameters (7,8). However, the

damaging effect of the exponential decaying electric impulsions, used by these authors, r e s u l t in a poor cell recovery. We report

experiments using a commercially available electropulsator

delivering short, voltage. A

square

permeability

"Lucifer Yellow"

(11,12)

predetermine the

best

shaped, assay

repeated, electric pulses of high (9,10)

proved settings

0006-291X/88 $1.50 Copyright © 1988 ~ Aca~mic Press, ~c. AH r ~ h ~ ~ reproduc~on ~ a ~ ~ r m rese~ed.

982

to of

with be the

the a

fluorescent reliable

electrical

test

shocks

dye to for

Vol. 151, No. 3, 1988

transfection

.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Transient e x p r e s s i o n o f

cells transfected genes could demonstrated

be by

with

viral

permanently the

HLA

DNA,

SV40 nuclear T antigen in the

was

integrated

conversion

obtained. in

the

The transfected cellular

DNA

as

of human ~ 2 m transfected Daudi

cells. Material

and Methods

Cell lines : 10F2 is a B-EBV + lympnoblasto[d cell line derived from normal human lymphocytes. Daudi is a B lymphoblasto[d Burkitt cell line (13). The Daudi cells are HLA class I negative due to a ~ 2 m gene mutation blocking its translation (14). 8166 is a T l~nnpho[d line transformed by infection with the HTLVI virus (15). Jurkatt is a T lymphoid line derived from a leukemic patient (16). K562 is a multipotent hemopo[etic cell line (17). Chemicals : Lucifer Yellow CH (Sigma) (LY), i0 mM stock in water,was used a t imM. Geneticin (G418 GIBCO) was dissolved i n RPMI. Electropermeation technique We have used the "Electropulsator" commercially available from BIOBLOCK (Strasbourg, France). The output voltage from this generator ranges between 0 and i000 volts. All the experiments have been performed with i00 microseconds shocks delivered at 1 hertz frequency. The voltage intensity and the repetition of the shocks were the only parameters to vary. Cell cultures were usually divided by half the day before shocking. Immediately before the electropermeation process, the cells were washed twice in RPMI without serum and chilled at +4"C. DNA, at the appropriate concentration, was added to the cells and incubated for 10mn at +4 ° C before shocking. 0, i ml of cell suspension adjusted to 107/ml were introduced between the two electrodes (2.2 mm apart) and the electric shocks delivered as indicated. After the shocks, cells were kept at +4°C for 5-i0 mn, then incubated at 37°C for 20 mn. The cells were subsequently diluted in serum containing medium and grown in culture. "Lucifer Yellow Permeation Assay" : Lucifer Yellow (LY) is a 457 daltons highly fluorescent non toxic molecule (ii) which, under physiological conditions, does not enter the cells. The opening of transient "pores" in the cell membranes caused by the electric shocks (9,10) allows the penetration of the LY fluorescent dye. Only live cells will retain the dye (12). The optimal electropermeation parameters, were established for each cell line using the "Lucifer Yellow Permeation Assay" performed in parallel with evaluation of the cell viability and survival. The percentage of cells with fluorescent cytoplasm was evaluated under UV light with a fluorescent microscope. Viability of the cell suspension was assessed 3h and 24h after shocking b y t r y p a n b l u e exclusion. Transient expression of the SV40 transfected DNA: The circular LP/pUCI3 plasmid containing the SV40 wild type viral genome cloned at the BamH 1 site of pUCI3 has been used. The transient expression of the nuclear T antigen of SV40 has been assayed a f t e r 7 2 hours by indirect immunofluorescence on fixed cells with an anti-T antigen serum from an SV40 induced tumor-bearing hamster. Permanent expression of the h u m a n ~ 2 microglobulin transfected DNA : The circular pEMBL9 plasmid containing a full length human ~ 2 microglobulin 14 kb genomic DNA fragment Sal I/Sma I (p~2m13) (18) has been used at the final concentration of 250 ~g/ml 6 Cotransfection with pSVTK Nee at 50 ~g/ml was performed (2 x I0 Daudi cells, 8 shocks, 1300 volts/cm). Culture of the transfected cells was performed with selection medium (geneticin 1 mg/ml, a dose highly toxic for the original Daudi cell line).

983

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HLA antigen surface expression indirect immunofluorescence using Cells were either simply analyzed of the most H L A positive cells)

on the or by

Daudi cells was evaluated by W6/32 monoclonal antibody. analyzed and sorted (collection using an EPICS-C (Coultronics).

Results The influence (% of

T

of voltage on short time expression of transfected DNA

antigen

percentage of

positive

cells),the

permeabilized

Permeation Assay")

viable cell recovery and the

cells (determined by the "Lucifer Yellow

was examined.

The results with 5 human cell lines

are summarized on table i. The concentration (50~g/ml)

i.e.

5~g

to 1600 volts/om, shocks is

per million cells. Varying the voltage from i000

we observed first that the voltage intensity of the

very critical,

range of

values

very close

to

the best results being obtained in a narrow

(Fig. i). Second,

voltage

Yellow Permeation voltage for

the optimal voltage conditions

the conditions inducing irreversible

and can vary with each individual the optimal

of DNA was kept constant

conditions

Assay".

Lucifer

As

Yellow

are

cellular damages

cell line tested (table i). Third, can be predetermined

by the "Lucifer

can be observed on figure i, the best permeation

correlates with the voltage

giving the best expression results with the SV40 plasmid. Experiments

were

also

electric impulsions.

performed

to establish the optimum number of

The results with 5 or i0 consecutive

shocks were

TABLE 1

TRANSFECTION OF

VARIOUS H U M A N C E L L LINES WITH THE ELECTROPERMEAT ION METHOD

(square waves, 100~s impulsions, 10 shc~lks at i Hertz) Name of the line

Lineage

Lucifer YellOw permeatic~ assay

SV40 cc~taining p 1 ~ - ! d

5Ouq/~ optimum voltage

permeabillzed cells

T positive cells (72h)

1200

2%

1400

3%

90%

1400

4%

96%

1400

8%

1OF2

B EBV Lymphocytes

1200

65%

Daudi

S Surkltt lymphocytas

1300

90%

8166

T HTLVT i y m p ~ e s

Jurkatt

T lymphocytes

1400

K562

Multlpot ent Hematopo~teic cells

1400

7

984

optlmt~n voltage

Vol. 151, No. 3, 1988

T SV4O % positive

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

% viable cells

k

""~~

100

4.

"",

LY

% viable cells

% positive

O0

10( 80 60

\

50

50

40 20

1/ 8()0

-

1000 1200 1400 1600

1000 12'00 14'00 1600

860

K562 hematopoietic cell line LY

TSV40

C

% )ositive

°,6 viable cells

\

I00 ..°~ .....

80

! 1

% viable cells

/ lO0

,100

\

D

% positive

t

60

!

50

50 40 20

J 800

I 1600 1200 1400 1600

660

1000 1200 1400 1600

IOF2 B EBV cell line [] [] % fluorescence positive cells a .....~ % surviving cells Figure i.

Determination of the parameters Influence of the voltage

of

eleetropermeation:

K562 cells (panels A and B) and 10F2 cells (panels C and D) were submitted to 5 square shaped electric impulsions of increasing voltage. Parallel experiments were performed with SV40 containing DNA, 50~g/ml, or Lucifer Yellow, imM, to examine the transfection efficiency (% of T antigen positive cells 72h after shocking, in panels A and C, or % of Lucifer Yellow permeabilized cells, panels B and D) and the viable cell recovery (% of viable cells assessed 24h after shocking b y t r y p a n b l u e exclusion).

compared.

One

example

consecutive square

impulsions

n u m b e r of

T

positive

shocks

%

versus

(9

with

K562 gave

cells the

is best

shown

o n figure 2. i0

r e s u l t s d o u b l i n g the

c e l l s c o m p a r e d to the r e s u l t s o b t a i n e d w i t h 5 4,5 % T a n t i g e n p o s i t i v e cells).

985

T h e s e results,

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TSV40

LY % positive

% positive

/ 100 90 80 70 60 50 40 30 20 10 1200

A

13oo

1400

B

1400

K562 hematopo[etie cell llne

Figure 2.

----7

5 shocks

m

I0 shocks

Determination of the parameters of optimum number of electric impulsions

electropermeation:

K562 cells were submitted to a variable number (5 or i0) of square shaped impulsions of increasing voltage. Parallel experiments were performed with LP/pUCI3 plasmid, 50~g/ml, and Lucifer Yellow, imM, to examine the transfection efficiency (% of T antigen positive cells 72h after shocking, panel A, or % of Lucifer Yellow permeabilized cells, panel B) and the viable cell recovery.

obtained with the K562 llne, were also obtained with the 4 other cell lines tested.

The 8166

T

lymphocyte

optimum concentration plasmid containing ~g/ml

(i

f i g u r e 3,

to the

20

cell of

the

llne

exogenous SV40

viral

has

been

DNA. DNA

The was

used to establish the concentration of the v a r i e d from i0 to 200

~ g of DNA p e r m i l l i o n cells). AS c a n be o b s e r v e d on f r a c t i o n of T p o s i t i v e c e l l s w a s e s s e n t i a l l y l i n e a r l y

r e l a t e d to the e x o g e n o u s D N A c o n c e n t r a t i o n .

S t a b l e p e r m a n e n t i n t e g r a t i o n and e x p r e s s i o n of e x o g e n o u s D N A c o u l d be obtained using

this

technique.

For instance, w e w e r e able to o b t a i n

986

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.=

14 13 12

LU >

11

I---

10

0

9 8

I-- ,,..,. I,..

7

0

6 5

> O9

4 3 2 1 10

25

50

100

200

[DNA] /~g/ml Transfection with increasing concentrations of exogenous DNA (8166 line, square waves, 100J/s impulsion, 8 shocks of 1400 volts at I Hertz) Determination of the parameters of electropermeation: optimum concentration of exogenous DNA

Figure 3 .

8166 cells were submitted to square shaped electric impulsions in the presence of an increasing concentration of LP/pUCI3 containing DNA. The % of T antigen positive cells was evaluated 72h after shocking.

the permanent conversion of the HLA class I negative B lymphoid cells Daudi into HLA positive ~2 microglobulin transfected Daudi cells. The Daudi

cells

resistance

to

Daudi

(Fig.

medium, 12

%

4).

together

Three

cells

After

w i t h a full the

functional,

gene c a r r y i n g

days a f t e r the t r a n s f e c t i o n ,

were

a

with

length,

4

detected

weeks

as s t r o n g l y

culture

period

the

3 % of

HLA class

I

in s e l e c t i o n

of the geneticin resistant fl2m transfected Daudi cells HLA

Strongly

was sorted

gene

Neomycine.

the t r a n s f e c t e d

were

cotransfected

microglobulin

human~2

Positive

were

out

transfected HLA

positive. by

using

positive

The the

most HLA positive cell population cell

Daudi

sorting

cells

apparatus.

The

~2m

remained thereafter 97 % HLA

positive. Discussion The e l e c t r o p o r a t i o n efficient.

The

procedure

described

"Electropulsator"

987

in t h i s p a p e r is s i m p l e

delivers

square

waves

and

electric

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a

a

h

A

B

L

um

a

b

b

a

C

D

a: O r i g i n a l Daudi c e l l l i n e b: Daudl c e l l s c r a n s f e c t e d w i t h human ~2 m i c r o g l o b u l i n

~ene

Figure 4. HLA conversion of Daudi cells Transient expression of HLA class I antigen was evaluated 3 days after the transfection (panel A). After a 4 weeks selection period with geneticin, the neomycin resistant surviving cells were examined for HLA antigen expression (panel B). The most HLA positive, neomycin resistant, transfected cells were sorted out with the cytofluorographic cell sorter. The resulting population was examined for the expression of HLA class I antigen. The same cell population examined after 6 months in culture is shown in panel D.

impulsions through cells to

a

DNA

repetitive

apparatus used

here,

containing cell suspension , exposing the

electric differs

deliver e x p o n e n t i a l d e c a y i n g Using our method, a) Voltage

the

transfection is cells in

the

transfection

previously described ones which

electric impulsions

The

importance

fact

that

unefficient

suspension

efficient voltage

from

Thus,

(7,8).

two electric parameters have been found critical:

setting:

results from

shocks.

are

range

if

and

of the

if

this

electric

voltage

the

voltage

is

too

parameter low

the

is too high, the

irreversibly damaged. As a consequence the is

quite

narrow.

In addition the optimum

voltage appears to be different for each individual cell line tested. The "Lucifer

Yellow

to predetermine i0 shocks

the

delivered

permeation assay" was found to be very helpful optimal

settings,

b)

The

number

of shocks:

at 1 Hertz frequency gave better results than 5

shocks. The transient

expression

of

the

SV40

plasmid

was on the average

around 3-4 % of positive cells when using 50 ~g/ml of plasmid DNA.

988

Vol. 151, No. 3, 1988

However,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transfection

DNA concentration. range of

DNA

efficiency

A

linear

concentration

one occasion).

Lower

sonicated

improve the

efficiency.

efficiency

increased plasmid

was observed over a wide

up to 200 ~g/ml of DNA (and 500 ~g/ml in DNA concentrations

salmon

sperm

DNA,

Linearization

but

were tested in the did

not

appear to

of the plasmid did not boost

either.

The successful

transfection of HLA negative Daudi cells with a 14 Kb

~ 2 microglobulin positive

with

relationship

plasmid

presence of

increased

gene

transfected

square shaped

conversion

Daudi

cells

indicates

that our method,

impulsions,

exogenous

can

selection technique

is

very

had

to

be

the

for

completed

It the

a given gene. cells,

into

also

HLA using

for

the

It indicates that large

cells.

critical

Daudi

efficient

(19).

expressing

transfected

is

DNA

penetrate

permanently

selection of resistance,

permanent

of

DNA

transfectants

their

electric

stable expression fragments of

and

shows

the

establishment

of

In our hands,

the

according

by an additional

that

to

neomycin

selection,

using

the cell sorter.

Finally,

comparing

decaying electric equivalent

in

the

square

impulsions

several efficient

membrane.

square

were more

has been

functions

applied

to

However,

create

shaped

labile,allowing

recover their

, we found the experimental

aspects.

generally more With

shaped impulsions to the exponential

the

transitory

impulsions,

former pores

the permeation

procedures method

on the cell structures

the cells to repare their membrane and viability

successfully

to

a

was

and to

in a shorter time. Our method wide

spectrum of nonadherent

cells as well as adherent lines.

References i. Graham, F.L., and van der Erb, A.J. 2. Mc Cutchan, J.H., and Pagano, J.S. 41, 3 5 1 - 3 5 5

989

(1973) Virology 52, 456-458 (1968) J. Natl. Cancer. Inst.

Vol. 151, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3. Chu, G., and Sharp, P. (1981) Gene 13, 197-201 4. Schaffner, W. (1980) Proc. Natl. Acad. Sci. USA 77, 2163-2169 5. Rassoulzadegan, M., Binctruy, B., and Cuzin, F. (1982) Nature 295, 257-259 6. Falkner, F.G. and Zachau, H.G. (1984) Nature 310, 71-74 7. Chu, G., Hayakawa, H., and Berg, P. (1987) Nuc. Acids Res. 15, 1311-1317 8. Knutson, J.C., and Yee, D. (1987) Analytical Biochemistry 164, 4449 9. Zimmermann, U. (1976) Biochim. Biophys. Acta 694, 227-231 i0. Loyter, A., Scangos, G.A. and Ruddle F.H. (1982) Proc. Natl. Acad. Sci. USA, 79, 422-426 ii. Stewart, W.W. (1978) Cell 14, 741-747 12. Mir, L., Banoun, H., and Paoletti, C. Exp. Cell. Res. In press 13. Klein, E., Klein, G., Nadkarni, J.S., Nadkarni, J.J., Wigzell, H., and Clifford, P. (1967) Cancer Res. 28, 1300-1305 14. Rosa, F., Berissi, H., Weissenbach, J., Maroteaux, L., Fellous, M., and Revel, M. (1983) EMBO Journal 2, 239-244 15. Salahuddin, S.Z., et al, (1983) Virology 129, 51-56 16. Gillis, S., and Watson, J. (1980) J. Exp. Med. 152, 1709-1716 17. Lozzio, C.B., and Lozzio, B.B. (1975) Blood 45, 321-326 18. Gussow, D., Rein, R., Ginjaar, !-, Hochstenbach, F., Seeman, G., Kottman, A. and Ploegh, H. (1987) J. Immunol. 139, 3132-3137 19. Fellous, M., Kamoun, M., Wiels, J., Dausset, J., Clements, G., Zeuthen, J., and Klein, G. (1977) Immunogenetics 5, 423-429

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