Gene, 81 (1989) 151-158

Elsevier

GENE 03102

151

Neutral selection of transfected mammalian cells using tissue plasminogen activator gene expression

(Recombinant DNA; genetic transformation; caseinolysis; proteolytic enzyme; SV40 and Moloney virus

promoters; expression vector)

Patricia Rogers”, Richard Fisher b and Jerry Guyden c

a Department of Microbiology and Immunology, University of California, Berkeley, CA 94720 (U.S.A.), Tel. (415)642-3148; b Biogen Research Corp., Cambridge, MA (U.S.A.), Tel. (617)864-8900, and’ Department of Biology, The City College of New York, New York, NY 10031 (U.S.A.)

Received by S.T. Case: 25 January 1989

Accepted: 10 March 1989

SUMMARY

The gene that produces the proteolytic enzyme, tissue plasminogen activator (tPA), was used as a selectable

marker for transfection. Cells that were successfully transfected with plasmids containing a gene for tPA (@a)

produce plaques in the caseinolysis assay. Two @a-containing plasmids were constructed and used to transfect

a variety of cell types. One plasmid contained the promoter region of simian virus 40 and the other contained

the Moloney murine leukemia virus promoter. No significant ditference in transfection frequencies was found

for the two plasmids. However, 80% of the cell types tested produced plaques. This system allows for the

identification and isolation of transfected cells under conditions that are not lethal to nontransfected cells.

INTRODUCTION

The development of procedures to transfer genes

into mammalian cells (Szybalska and Szybalski,

1962) in a way that facilitates their expression has

Correspondence to: Dr. J. Guyden, Department of Biology, The

City College of New York, New York, NY 10031 (U.S.A.)

Tel. (212)690-8449; Fax 690-6696.

Abbreviations: A,,,, absorbance at 600 nm; Ap, ampicillin;

cDNA, DNA complementary to RNA; Cm, chloramphenicol;

FCS, fetal calf serum; HAT medium, hypoxanthine-aminopterin-

thymidine selective medium (Szybalska and Szybalski, 1962);

HGPRT, hypoxanthine-guanine phosphoribosyl transferase;

LB, Luria-Bertani medium; LTR, long terminal repeat;

MoMuLV, Moloney murine leukemia virus; PEG, polyethylene

glycol; SV40, simian virus 40; tPA, tissue plasminogen activator;

tpa, gene (DNA) coding for tPA.

revolutionized the way we study gene regulation. The

most popular method used to facilitate gene transfer

is Ca. phosphate precipitation (Graham and Van

der Eb, 1973; Mulligan and Berg, 1980), although

other techniques such as protoplast fusion (Oi et al.,

1983) and electroporation (Neumann et al., 1982)

have been reported to yield improved transfection

frequencies, especially in cells that grow in sus-

pension. Cells that are successfully transfected using

either of these methods are currently selected under

conditions that result in cell death if the implanted

gene is not expressed. For example, HGPRT-

negative cells will not survive in HAT media unless

stable incorporation and expression of an exogenous

HGPRT gene is facilitated through transfection or

cell hybrid formation (Szybalska and Szybalski,

1962). Experimentally, these techniques only allow

0378-I 119/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

152

for the isolation of stable transfectants that constitu-

tively express marker genes at levels that overcome

the restrictive conditions. Transfected cells are killed

if expression of the selected gene is transient or below

that level required to survive the selective challenge.

Consequently, such transfected cells are not

detected. In this paper we describe the development

of a transfection system that allows the identification

and analysis of the expression of transfected genes

using a neutral selection system. That is, the trans-

fected subpopulation is identifiable under conditions

that do not cause death to cells that did not receive

DNA. In this system, successful transfection is

obtained by the transfer and expression of the tpa

gene. TPA expression in a single cell can be detected

using these assay conditions, and unlike other sys-

tems (Antin et al., 1988), the transfected cell remains

viable and may be recovered.

TPA is one of a series of enzymes necessary for

fibrinolysis (Tilsner and Lenau, 1980). A full length

cDNA and a portion of the genomic promoter for tpa

have recently been cloned (Fisher et al., 1985; Penni-

ca et al., 1983). The advantages of using this gene as

a selectable marker for transfection are: (1) the tpa

gene product is released from cells in culture allowing

the assay of intact cells; (2) single cell transfectants

can be identified and cloned which may facilitate the

study of the molecular relationship between transient

and stable transfection and (3) the detection assay

for the expression of the tpa gene, caseinolysis, is

very inexpensive and relatively easy to perform

(Taylor et al., 1972). Caseinolysis involves the

plating of cells in a casein-agarose mixture. TPA is

released from expressing cells and activates the en-

zyme cascade that degrades the casein surrounding

the cell to form a visible plaque. The cell is visible in

the middle of the plaque using a light microscope and

cell viability is preserved because the casein-agarose

mixture contains normal tissue culture ingredients.

The caseinolysis system has been exploited here to

test for the expression of tpu in plasmids containing

promoters from either SV40 or MoMuLV. Optimal

conditions were developed for delivery of both tpu -containing plasmids into protoplasts.

MATERIALS AND METHODS

(a) Cells

All cell lines were maintained in RPM11640

medium supplemented with 10% FCS (Sigma, St.

Louis, MO), 1 mM Na. pyruvate, 2 mM L-

glutamine, and 50 pg/ml of gentamycin sulfate

(Gibco, Grand Island, NY) at 37°C. Cells were split

the day prior to transfection to insure logarithmic

populations. The murine cell lines used were: K46R

(mature B cell line), AGX5863 (myeloma), (both

gifts of M. Koshland, University of California,

Berkeley, CA), J558L (myeloma) and SP2/0 (hybrid-

oma) (both obtained from V. Oi, Becton-Dickinson,

Mt. View, CA) from the B cell lineage, and BW5 147,

EL4, E1.17, and C6VL (all are T lymphomas

obtained from J.P. Allison, University of California,

Berkeley, CA) from the T cell lineage. The human

monocyte line HL60 was purchased from American

Type Culture Collection, Rockville, MD.

(b) Plasmids

The pTPA114 plasmid was cloned by R. Fisher

(Fisher et al., 1985) Biogen Research Co. This plas-

mid contains the 5’-flanking region of the genomic

human tpu gene and the complete cDNA for human

tpu cloned into pUC8 vector sequences. pL1, a

cloning vector that possesses the pBR322 origin of

replication, the Ap-resistance gene, and the SV40

promoter were obtained from M. Koshland, Univer-

sity of California, Berkeley, CA. pMSD containing

the same bacterial sequences as pL1, the MoMuLV

3’ long terminal repeat sequences (LTR), and the

SV40 16s and 19s donor and acceptor sites and

polyadenylation signal were a gift from Tom

St. John, University of Washington, Seattle, WA. In

the pMSD/? construct, the murine T cell antigen

receptor b-chain gene has been inserted 3’ to the

MoMuLV sequences by J. Allison. All plasmids

were transformed into the Escherichiu coli bacterial

strain MC1061 (Clontech, Palo Alto, CA).

(c) Protoplast preparation

Large single colonies of E. cofi strain MC1061

from freshly streaked LB/Ap plates were added to

25 ml of LB containing 50 pg Ap/ml and grown to an

153

~0.5 kb

Fig. 1. Construction of the rpa carrying plasmids. (Panel A) A

3.15-kb BamHI fragment which contains the complete cDNA for

human tissue type plasminogen activator (but lacking any ge-

A 600 = 0.6 with shaking at 37°C. The cultures were

incubated further for 16-18 h at 37’ C in the presence

of Cm to amplify plasmid copy number. To equalize

the number of starting bacteria for every batch, a

culture volume equivalent to 25 ml of bacteria at an

A 6oo = 0.6 was centrifuged (2500 x g, 15 min, 4°C).

Protoplasts were prepared as previously described

by Oi et al. (1983). Briefly, for every 25 ml culture,

1.25 ml of chilled 20% sucrose/O.05 M Tris. HCl

pH 8 was added to the pellet, and cells were resus-

pended by vortexing. 250 ~1 of a 5 mg/ml solution of

lysozyme in 0.25 M Tris * HCl pH 8, was incubated

with the bacteria for 5 min on ice, and 0.5 ml of cold

0.25 M EDTA pH 8, was added to the mixture for

a further 5 min incubation. After the addition of

0.5 ml of Tris . HCl pH 8, the bacteria were trans-

ferred to a 37°C water bath and incubated for

10 min. The resulting protoplasts were diluted slowly

with swirling with 10ml warm RPMI1640/10%

sucrose/O.01 M MgCl,, and incubated 10 min at

25°C. The preparation was examined using a 20 x

objective of an inverted microscope with phase con-

trast optics to confirm conversion of the bacteria to

protoplasts, and was used immediately for fusion.

The amount of DNA contained in the protoplasts

was determined by comparison to known concentra-

tions of DNA standards (a Hind111 digest of bac-

teriophage 1 DNA) after electrophoresis in a 0.6%

agarose gel at 60 V for 5 h. The amount of DNA in

the gel was used to calculate the amount of DNA per

individual protoplast and the amount of DNA

delivered in a typical fusion experiment. The number

of bacteria/ml was determined by streaking 1~1 of

several dilutions of overnight cultures (A60o = 0.6)

on LB/Ap plates, incubating overnight at 37’ C, and

counting the number of distinct colonies.

nomic rpa promoter sequences) and the SV40 small t antigen

polyadenylation signal was removed from pTPAll4 and ligated

into the unique BumHI site of pL1. This placement positioned

the rpa gene immediately 3’ to the SV40 early region promoter.

The resulting plasmid was named pSVTPA and used in the

construction of pMTPA shown in panel B). To place ipu gene

expression under the control of the MoMuLv LTR, a 0.62-kb

HindHI-XhoI fragment of the MSD/I plasmid containing the

MoMuLV promoter was inserted between the Hind111 and XhoI

sites of the pSVTPA plasmid after removing the SV40 promoter.

Abbreviations for restriction enzymes: B, BumHI; Bg, BglII; C,

&I; E, EcoRI; H, HindHI; S, SsrI; X, XhoI, Amp’=

Ap-resistant.

154

RESULTS AND DISCUSSION ,b) Characteristics of protoplasts

(a) Plasmid construction

The plasmid pTPAll4 contains pUC8 vector se-

quences, the genomic 5’-flanking region and the

complete cDNA for the human tpa gene. To prepare

for cloning, the plasmid was transformed into

MC1061 cells, amplified and mapped. The se-

quences coding for TPA and the SV40 small t anti-

gen polyadenylation signal were contained in a

3.15kb BumHI cassette. These sequences were iso-

lated and ligated into the unique BarnHI site of pL1

(Fig. lA), directly adjacent to the SV40 early region

promoter and the 19s splice acceptor site. Com-

petent MC1061 bacterial cells were transformed

with this construct and positive colonies were identi-

fied using a 2.0-kb BgZII fragment located within the

tpa BumHI cassette. Clones in which the BumHI

cassette was inserted in the proper orientation in

relation to the SV40 promoter were isolated and

termed pSVTPA. A second expression vector was

created in which the SV40 promoter was replaced by

promoter sequences from MoMuLV. Plasmid DNA

from pSVTPA was digested with Hind111 + X/z01

and the resulting 6.22-kb fragment, containing all of

the pSVTPA sequences except the SV40 early region

promoter and the origin of replication, was ligated to

a 0.6-kb Hind111 + X/z01 fragment from pMSD-/I, a

plasmid that contains the 3’ LTR from MoMuLV

(Fig. 1B). This plasmid was termed pMTPA.

TABLE I

Quantitation of plasmid DNA in protoplast

Plasmids a Number of bacteria b

(per 0.8 ml)

Plasmid DNA ’

@g/O.8 ml protoplast)

Plasmid DNA d

(pg/protoplast)

pMTPA

pSVTPA

6.4 x 10’ 2.625 4.1 x 10-s

9.6 x IO’ 0.722 8.0 x 10m9

a Cells of E. cofi strain MC1061 were transformed with the plasmids pMTPA and pSVTPA, diluted and streaked onto LB/Ap plates

and grown at 37°C.

’ Colonies were counted and corrected for the dilution factor to obtain the number of bacteria per 0.8 ml.

’ For DNA quantitation, bacteria from a single colony were grown overnight in the presence of 200 ng Cm/ml and the volume of the

culture was adjusted to obtain an A 6,,,, = 0.6. 1.5 ml of the culture was used to isolate plasmid DNA, which was subjected to

electrophoresis (estimates of DNA concentrations were made based on the intensity of the plasmid bands versus known amounts of

the DNA standards Hind111 digested I DNA).

d The number obtained in c divided by the number obtained in b.

The use of protoplast fusion to mammalian cells

(Oi et al., 1983) provides an alternative method of

plasmid transduction. Both of the @z-containing

plasmids were used to transform cells of the bacterial

strain MC1061. Clones of each type were isolated

and used to quantitate the number of bacteria/ml and

the amount of plasmid DNA/protoplast. The results

presented in Table I show similar cell growth quan-

tities/ml of bacteria for both pMTPA and pSVTPA,

but pMTPA produces over live times the amount of

plasmid DNA/protoplast as does pSVTPA. Based

on a weight of approx. 8.66 x lo- i2 pg/plasmid, the

plasmid copy number/bacterium was calculated to

be 4767 for pMTPA and 930 for pSVTPA using the

following formula: the number of base pairs for the

plasmid is multiplied by the average nt weight divided

by Avogadro’s number, to obtain the weight per mol.

This number is then multiplied by 2 because the

plasmid is double-stranded. In an attempt to deter-

mine the plasmid DNA concentration that produces

maximal transfection frequencies, increasing vol-

umes of each plasmid preparation were fused to

3 x lo6 J558L cells (a myeloma cell line) and assay-

ed using caseinolysis (Fig. 2). The results of this

experiment show optimal conditions to be a function

of the volume of protoplast rather than plasmid con-

centration. A volume of 0.8 ml of each protoplast

type produced the largest number of plaques and was

used in the remaining experiments.

Transfection vs Volume of Protoplasts

0.2

0.1

0.0 0 1 2 3

Volume of Protoplasts

Fig. 2. Transfection versus volume of protoplast. Various

volumes of protoplasts made from the bacterial strain MC1061

(equalized to an A,,, = 0.6) were fused to 3 x IO6 J558L cells as

described by Oi et al. (1983). The volumes of protoplasts used

were 0.3, 0.8, 1.2 and 2.0 ml. Triplicate samples of the treated

cells were plated in the casein-agarose mixture and incubated at

37°C for 24 h. The transfection frequency was calculated based

on the number of plaques counted per total number of viable

cells. Each time point is the average of three plates. (0) Cells

fused to pMTPA-carrying protoplasts. (+) Cells fused to

pSVTPA-carrying protoplasts. (m) Cells fused to the control

plasmid pL1.

(c) Caseinolysis

An example of the plaques produced by cells

transfected with both pMTPA and pSVTPA is

shown in Fig. 3. Plaques are visible 2 h post fusion

(Fig. 4) and the number of plaques increases to 4 h

but then remains constant for up to 24 h. The size of

the plaques continues to increase but the final size

and time of expansion is highly variable between

individual plaques (data not shown). Plaque size

ranged from 0.1-2.0 mm in diameter and no differ-

ence in average size was detected between pMTPA-

and pSVTPA-induced plaques. When transfected

cells are plated at various times after PEG fusion

(Fig. 5), the maximum number of plaques are pro-

duced in cells plated at 4 h. The number of plaques

drastically decreases with time in plates seeded after

4 h. However, when cells are plated between 13 and

24 h after exposure to PEG, another increase is de-

tected and even a small number of plaques is pro-

duced in 42-h platings.

The efficiency of transfection by both pSVTPA

and pMTPA was tested in a variety of TPA negative

Fig. 3. TPA caseinolysis on the casein agarose plates. J558L

cells were prepared as described in Fig. 2 and plated 4 h after

fusion in 100 mm tissue culture dishes. The pictures were taken

48 h after plating. (A) Control pll-transfected cells.

(B) pMTPA-transfected cells. (C) pSVTPA-transfected cells.

Viable fused cells (3 x 106) were added to 4 ml of prewarmed

(37 o C) casein-agarose mixture in a sterile 15 ml centrifuge tube

(Beckton Dickinson, Oxnard, CA). The mixture consisted of

10% acid-treated FCS, 1.9% Carnation nonfat dry milk, and 1%

standard agarose (Bio-Rad, Richmond, CA) in RPM11640

containing 2 mM L-glutamine and 1% gentamycin. The tube was

inverted for mixing and the contents were poured into 100 mm

tissue culture dishes (Becton Dickinson). The plates were

allowed to harden at room temperature for 15-30 min and

incubated at 37°C for 24-48 h before analysis.

Transfection Frequency vs.Time

I- O.‘- 7 o.ojll___

0 10 20 30

Time ( Hours )

Fig. 4. Transfection frequency vs. time. J558L cells were fused to

0.8 ml ofprotoplasts made from the bacterial strain MC1061, as

described in Fig. 2, and plated in triplicate for each time point.

The number of plaques per plate was determined at 1 through 6

and 24 h for cells fused to pMTPA (0) or pSVTPA (+). No

plaques were detected in pL1 transfected cells. n represents the

combination of 0 and +, where they overlap.

156

Transfection Efficiency vs.Time of Plating

d lb 2b 30 40 50

Time of Plating ( Hours )

Fig. 5. Transfection efficiency vs. time of plating. J558L cells

were plated in the caseinolysis assay, as described in Fig. 2, at

1, 4, 8, 12, 24, 28 and 42 h after fusion to pMTPA carrying

protoplasts (o), or pSVTPA carrying protoplasts (+). Each

time point represents the average number of plaques obtained

from three plates. The number of plaques per plate was deter-

mined 24 h after plating the cells. No plaques were detected in

pL1 transfected cells.

TABLE II

Protoplast-mediated transfection efficiency of ma-carrying plas-

mids

Cell line” % cells expressing plaquesb

J558L

SP20

K46R

BW5147

EL4

AgX5863

27.44

El.17

C6VL

HL60

pMTPA pSVTPA

0.286

0.187

0.00

0.030

0.005

0.005

0.006

0.017

0.00

0.126

0.100

0.00

0.013

0.010

0.003

0.002

0.012

0.00

0.002

a See MATERIALS AND METHODS, section a, for a de-

scription of each cell line.

b Approx. 3 x lo6 cells were transfected with 0.8 ml of proto-

plasts as described in Fig. 2. Cells were counted and plated in

triplicate in the casein-agarose 4 h after fusion. Plaques were

determined 24 h after transfection. The percentages were calcu-

lated by dividing the number of plaques by the number of plated

cells. All of the numbers presented in the table, except those for

J558L, represent an average of 2-5 trials. The numbers

presented for J558L cells represent an average of 22-25 trials.

None of the cells produced plaques when transfected with pL1.

cell lines (Table II). Of all the transfected cell lines

80% produced plaques with frequencies between

10 - 4-0.3 % . No plaques were found in the cell lines

C6VL nor K46R in three separate trials, while about

0.3% of transfected J558L cells produced plaques.

No significant difference in transfection frequency

between the two plasmids was detected within any

cell line tested. A twofold higher number of plaques

was found for pSVTPA over pMTPA in the cell line

EL4, although the majority of cell lines produced

more plaques when transfected with the pMTPA

plasmid.

The advantages of using this detection system are;

(I ) the ease of performance, inexpensive nature, and

rapid development of the procedure (the assay uses

‘Carnation’ nonfat instant milk and successful trans-

fection can be determined only hours after plating);

(2) the ability to identify individual cells within a

treated population that are successfully transfected;

and (3) the use of nontoxic assay conditions with the

potential for the recovery of transfected cell for

further study. One limitation of using tPA as a

marker for transfection is the requirement that the

target cells not express caseinolytic products. During

the development of our system we discovered that

the following lines release endogenous caseinolytic

products and cannot be used in this assay system:

S107 (myeloma), HS27 (human libroblast), 231R

(lymphoma), 702 (pre-B cell), P8 15 (murine masto-

cytoma), and NIH3T3 (mouse fibroblast). Normal

mouse spleen cells were also found to be highly

caseinolytic, although sorted or purified splenic sub-

populations have not yet been tested.

Several factors were critical for obtaining optimal

tPA expression in the caseinolytic assay. First, large

bacterial colonies from freshly-streaked plates pro-

duced higher percentages of transfectants. Addition-

ally, it was critical to verify, microscopically, a com-

plete shift of bacteria to protoplast after lysozyme

treatment. Inadequate lysozyme treatment resulted

in bacterial contamination and over-treatment great-

ly reduced the transfection frequency. Other at-

tempts to improve the transfection frequency includ-

ed increasing the protoplast-to-cell ratio during

fusion in an effort to deliver more plasmid DNA to

target cells, since about 10% of all transfected plas-

mid DNA is mutated by the recipient cell (Calos

et al., 1983). It appeared, however, from the results

described in Fig. 2 that the amount of plasmid DNA

available for transfer to target cells was limited by protoplast concentration. That is, as the number of bacteria for both plasmids at A,,, = 0.6 is similar, it is possible that the number of protoplast in 0.8 ml (2-9 x 10’) represents a saturating concentration for available fusion sites on the surface of the target cell. (For 3 x lo6 cells there are 7-30 protoplast per cell in 0.8 ml). Increased numbers or volumes of protoplast above that present in 0.8 ml may sterically interfere with the fusion process. Overall, more pMTPA transfected cell lines produced plaques than the SV40-based plasmid, but the effect seemed to be totally dependent on the cell line rather than the type of promoter (Table II). This tendency could also be a function of the larger plasmid copy number/proto- plast of pMTPA (5 times as much as pSVTPA). Similar studies (Laimans et al., 1982; Levinson et al., 1982) show the SV40 enhancers to be much more active than MoMuLV tandem repeats in cells of simian origin. However, our studies dealt with the entire SV40 promoter region instead of isolated en- hancer elements, and it is possible that any advan- tage of the SV40 enhancer was masked by the rela- tive activity of separate regions of the promoter. In fact, it has been suggested that all of the sequence motifs within the SV40 enhancer may have different activities in different cell lines (Maniatis et al., 1987). Further studies of transcriptional activity in individ- ual monkey cells transfected with either pSVTPA or pMTPA would be a more precise measure of pro- moter strength because the level of protein trans- lation and RNA transcription do not always agree (Khoury et al., 1987). Our studies solely examine the expression of tPA.

Our caseinolytic assay has the potential to allow for the isolation of successfully transfected cells for growth and further analysis. Cells identified under caseinolytic plaques are effectively cloned. These cells can be removed from the agarose mixture with a drawn Pasteur pipet and suction, and grown to confluency. We are presently using this system to analyze the molecular basis for conversion of transient to stable expression in individually trans- fected isolates. Additionally, we are subcloning a second gene into both of the plasmids described here to study co-transfection using our system.

(d) Conclusions

(1) We have constructed plasmid vectors con- taining the tpa gene for the easy detection of trans- fected mammalian cells.

(2) The plasmids were delivered into several types of mammalian cells using protoplast fusion and successful transfection was determined by caseinoly- sis.

(3) The frequency of transfection using this selection system was high (between 10 - 4-0.3 %), especially for cells that grow in suspension and 80% of all cell lines tested produced plaques.

(4) We conclude that we have developed a trans- fection system that may offer several advantages to systems that use toxic assay conditions such as the selection of HGPRT-transfected cells in HAT media. A single cell transfected with either of our tPA-containing plasmids remains viable and can be isolated under nontoxic assay conditions.

ACKNOWLEDGEMENTS

We thank Dr. Leon Wofsy for his valuable support and helpful comments on this manuscript. This work was supported by U.S.P.H.S. grants CA 24436 and CA 9179, RCMI grant RR-03060, and NSF grant DCB-8714987.

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