Efficient transfection of mammalian cells with viral DNA in optimal culture conditions

Journal (if‘ Yimlogical ~t,t~rods, 7 (1983) 321-326

321

Elsevier JVM 281

EFFICIENT

TRANSFECTION

IN OPTIMAL

Standard efficiency Optimal

CULTURE

or newly developed

results

DNA

transfection

have been obtained

by treating

with DNA-calcium

phosphate

DNA transfection

procedures

virus 40 (SV40) DNA and monkey

using simian

before infection

OF MAMMALIAN

sv 40

CELLS

WITH

VIRAL DNA

CONDITIONS

the delicate

were compared

kidney

for both

innocuity

(Vero) cells as an indicator

cell monolayers

with a solution

and

system,

of glycerol

complexes.

Vero cells

INTRODUCTION

The introduction of foreign DNA into a cell represents a powerful tool for understanding gene expression and cell transformation at the molecular level. A number of transfection procedures have been developed to circumvent the inefficient uptake of exogenous DNA by mammalian cells (McCutchan and Pagano, 1968;Graham and Van der Eb, 1973; Cooper and Silverman, 1978; Copeland and Cooper, 1979; Stow and Wilkie, 1976; Long et al., 1980). Unfortunately, most of these procedures destroy a proportion of the delicate cell monolayer. In this study, we describe a simple and efficient DNA transfection technique which maintains sensitive cells in optimal culture conditions. MATERl.4LS

AND METHODS

Cell.r and viral DNA

African green monkey kidney cells (Vero) were grown in Eagle’s minimal essential medium (MEM) supplemented with lOye fetal calf serum (FCS) and 50 &ml gentamicin. Simian virus 40 (SV40) DNA was purified by both phenol and chloroformisoamyl alcohol (24: I) extraction of the Hirt supernatant, followed by banding in a ethidium bromide-cesium chloride gradient (Hamelin and Yaniv, 1979). ‘To whom correspondence Olh6-0924/Xi/S03.00

should

be addressed.

5’ 1983 Elsevier

Science Publishers

B.V

322

Transfe~tion procedures Ceil monolayers in 25cm2 plastic flasks were overiaid with 1 ml of SV40 DNA (1 ug/ml) in HEPES-buffered saline (Wigler et al., 1977) in the presence or absence of 0.125 M CaCl, (Graham

and Van der Eb, 1973). Six different

were used: (a) cells were left in contact with the DNA solution

transfection

procedures

for 30 min at 37°C before

addition of MEM supplemented with 2% FCS; (b) cells were treated with 20% glycerol in HEPES buffer for lo- 15 set before addition of the viral DNA; (c)cells were exposed to 20% glycerol for the same period of time after incubation with DNA (Copeland and Cooper, 1979); (d) cells were treated with 25% DMSO in HEPES buffer to IO-15 set folowing transfection (Stow and Wilkie, 1976); (e) cells were preincubated for 1 h at 37°C in hypertonic (0.6 M NaCl) culture medium before addition of DNA (Long et al., 1980); and (f) cells were incubated for 30 min at 37°C in Tris-bufferedculture medium without serum, containing 0.5 mglmi DEAE-dextran (MW 50,000) and the viral DNA (McCutchan and Pagano, 1968). Transfected cells were maintained at 37°C in MEM with 2% FCS until the appearance of cytopathic effects. Cellular uptake of viral DNA Immediately after transfection with 3H-labeled SV40 DNA (112,000 cpm/pg), the cells were monodispersed with trypsin, washed three times with isotonic buffer and collected by filtration onto Whatman GF/C filter discs. Discs were washed twice with 5% trichloroacetic acid, twice with ethanol, dried and counted in aliquid scintillation spectrophotometer. In some cases, transfected cells were treated with 50 ug/ml DNase for 30 min at 37’C before trypsinization and counting to evaluate the amount of DNA adsorbed to the cells. RESULTS

AND DISCUSSION

An increasing number of studies in molecular biology rely on the permeability mammalian cells and the introduction of exogenous nucleic acids. The ability

of of

mammalian cells to take up exogenously added DNA and to express genes included on that DNA critically depends, however, on the particular cell line used as the recipient (Stow and Wilkie, 1976; Graf et al., 1979; Milman and Herzberg, 1981; Scangos and Ruddle, 1981; Shen et al., 1982). In our laboratory, we have been interested in the detection of long-term phenotypic changes caused by specific viral genes introduced into a variety of mammalian cells. Usual DNA transfection procedures soon appeared as more damaging to a number of cell lines than to others. Monkey cell (BSC-1, MA, Vero) monolayers, for example, were more rapidly destroyed at 37°C after exposure to various chemicals than their murine (L, 3T3, 3T6) or human (MRC-5, IAFP-1, WI-38) counterparts (unpubl. results). Thus, it seemed important to find the most appropriate method for introducing DNA into a variety of mammalian cells in culture. DNA transfection procedures already described in the literature (McCutchan and

Pagano,

1968; Graham

and Van der Eb, 1973; Cooper and Silverman,

1978; Copeland

and Cooper, 1979) or developed in our laboratory, were compared for both innocuity and efficiency using SV40 DNA and Vero cells as an indicator system. Figure 1 shows cells transfected

with viral DNA

according

to the different

procedures.

Vero cell

monolayers pretreated with glycerol (Fig. lb) or DEAE dextran (Fig. If) were similar to controls (Fig. la). Significant cell losses were observed, however, when glycerol or DMSO was added immediately after DNA transfection (Fig. lc, d). Pretreatment of

Fig.

1. Vera

(c) glycerol pretreatment. stalned

with

cells

transfected

with

post-trearment: At

the end

SV4O

(d) DMSO

DNA

of transfeotion.

hemato~ylln-eosin.

200

in theahsrnca

post-treatment; cell X.

monolayers

ofCaC1,.

(e) sodium were

(a)control:

chloride rimed

with

(b)glycerol

pretreatment;

pretreatment;

(f) DEAE

phosphate-buffered

dextran saline

and

324

the cell monolayer with sodium chloride (Fig. le) led to its nearly complete destruction. A slightly higher uptake of 3H-labeled SV40 DNA by Vero cells was noted only for the DMSO

and the high salt procedures

(Table

1).

Coprecipitation of the viral DNA with calcium phosphate significantly increased the efficiency of transfection (Table 1) but damage to the cell monolayer was generally more pronounced (Fig. 2). A high level of degeneration with important cell losses were effectively observed in the control (Fig. 2a) as well as in the cell monolayers treated with glycerol (Fig. 2c) or DMSO (Fig. 2d) after inoculation of DNA. Very little

Fig. 2. Vero cells transfected Fig. 1.

with SV40 in thepresence

of CaCI,. The different

treatments

are lettered as in

325

TABLE Uptake

I of SV40 DNA by Vero cells before and after different Control

DNA DNA+CaCI, DNA+CaCI,+DNase “Cells were transfected

891b

pre- or post-treatments”

Glycerol

Glycerol

pre-treatment

post-treatment

DMSO

Sodium

DEAE

chloride

dcxtran

787

426

1,708

2,544

579

102,566

83,740

11,351

8.855

16,820

65,594

82,053

60,293

9,535

6,163

15,088

53,788

in 25-cm’ culture

flasks.

with

1 pg of 3H-labelled

’ Results given in cpm. Average

viral DNA (112,000 cpnw’pg)

of five experiments.

remained of the cells incubated with hypertonic medium before transfection (Fig. 2e). Only cells pretreated with glycerol (Fig. 2b) or DEAE-dextran (Fig. 2e) appeared intact. The amount of DNA found in association with glycerol-pretreated cells was also intermediate between that of control and DEAE dextran-pretreated cells (Table 1). More than 70% of the input radioactivity remained associated with control or treated cells after washing and DNase treatment (Table 1). Non-specific adsorption of DNA on cell membrane was thus minimal in all cases. SV40particles were observed by electron microscopy in transfected cells as well as in tissue culture fluids after 3 to 5 days of incubation at 37°C. Maximum infectivity of viral DNA, measured by mean tissue culture infective doses (TCID,J or by plaque-forming units (PFU), was obtained with the glycerol pretreatment technique (results not shown). Problems related to the destruction of the cell monolayersfollowing DNA transfection have been neglected in the past but may now represent a major handicap as more sensitivity is progressively needed in tests. Rare events such as those assayed in transformation or recombination studies may be detected by our modification of the standard calcium phosphate technique which offers high transfection efficiency together with optimal cell conservation. Simple and rapid to perform, the glycerol pretreatment technique has already been used in our laboratory to induce persistent infections and transformation of Vero cells with supercoiled, relaxed or linear SV40 DNA (unpubl. results). We are now working with more complex DNA molecules such as rccolnbinant plasmids containing fragments of human cytomegalovirus (HCMV) genome to confirm the practical advantages of our transfection technique in genetic mapping by marker rescue. The expression of cloned HCMV genes in different types of mammalian cells is also being studied. ACKNOWLEDGEMENTS

This research was supported in part by the Conseil de la recherche en Sante de Quebec, the Cancer Research Society Inc., and by funds from the institut Armand-

326

Frappier. &e

One of us (J.Y.) acknowledges

de I’Education

du Quibec

post-graduate

and the Cancer

scholarships

Research

Society

from the MinisInc.

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