Vol. 3, 127-134, February 1992 Cell Growth & Differentiation 127

Inducible Transformation of Cells from Transgenic MiceExpressing SV4O under lac Operon Control’

Ruth Epstein-Baak,2 Ying Lin, Vidoria Bradshaw, andMelvin CohnThe Salk Institute, Developmental Biology Laboratory,

San Diego, California 92 186-5800

AbstractIf it were possible to clone in vitro cells of any type, atany stage of differentiation, from an extensivelycharacterized animal such as the mouse, many areas ofcell biology would benefit. Indeed, it would be evenmore helpful if these cells could subsequently berestored to their normal in vivo phenotype wheneverrequired. Here, we describe a step on the pathway tosuch an idealized “clonable” mouse. In principle, itseeks to link a “universal” transforming agent to aregulatory system that is relatively simple, yet quiteforeign to the mouse. A plasmid containing thebacterial lac operator/promoter region linked to theSV4O large T antigen and a vedor containing the Iacrepressor that can be expressed in mammalian cellswere coinjected into fertilized mouse oocytes utilizingthe standard techniques for generating transgenic mice.Two progeny were obtained that express large Tantigen in the presence, but not the absence, of thenonmetabolizable lac inducer, isopropyl-/3-thio-D-galactoside. This report characterizes fibroblast celllines established from these transgenics that are readilytransformed in vitro with isopropyl-fl-thio-D-galactoside. A significant proportion of the cells arerestored to their “normal” (nontransformed phenotype)when isopropyl-fl-thio-D-galdctoside is removed.

Introduction

In this study, the SV4O TAg3 was utilized as the prototypetransforming gene, as its expression has been widelystudied in transgenic mice with the objective of under-standing the progression from oncogene expression andcellular hyperplasia to tumor formation. In previous stud-ies, the SV4O transgene was regulated either by its own(1 -3) or by an exogenous promoter (4-9). In two in-

Received 9/16/91.1 Supported by NIH Allergy and Infectious Disease Grant ROl A105875(R. E-B., M. C.) and NIH Training Grant CA 09256 (V. L., V. B.).2 To whom requests for reprints should be addressed, at the Salk Institute,Developmental Biology Laboratory, P. 0. Box 85800, San Diego, CA92186-5800.3 The abbreviations used are: 5V40 TAg, simian virus 40 large T antigen;

FCS, fetal calf serum; IPTC, isopropyl-fl-thio-D-galactoside; RSV, Roussarcoma virus; PBS, phosphate-buffered saline; PBSAE, PBS with sodiumazide and ethylenediaminetetraacetate (EDTA); ts, temperature sensitive;PAGE, polyacrylamide gel electrophoresis; 1#{176},cells which have neverbeen exposed to IPTG; l�, cells continually grown in the presence ofPIG; l�-�I, l� cells from which IPTC has been removed; I0��.l+, de novo

addition of IPTG to cells which had never been exposed to IPTG prior tothe time of addition of inducer.

stances, the fusion plasmids incorporated reversible reg-ulatory systems which were then utilized to make trans-genic animals. The first example placed SV4O TAg underthe control of a divalent cation inducer (zinc or cadmium)by fusion with a plasmid containing a thymidmne kinasegene that was controlled by a metallothionein promoter(1). The second case used a thermolabile ts mutant ofTAg (tsA58), placed under the control of the H�2Kbpromoter, which can be further induced by interferons(10). The tsA58 TAg can conditionally immortalize tissueat the permissive temperature (33#{176}C),which is, however,a suboptimal temperature for growth of most mammaliancells. The unique feature of the transgenic mice de-scribed in this report is the presence of a completelyforeign regulatory system, which is efficiently responsiveand easy to manipulate. Utilizing this regulatory system,we have demonstrated the reversible expression of TAgas a function of the exogenous inducer, IPTG.

Results

Description of Transgenic Lines. Transgenic mice wereobtained by coinjecting the pRSV-1 (11) and pSVlacO(12) plasmids. pRSV-1, the kind gift of M. C-T. Hu,contains the lad repressor, whereas pSVlacO, generouslydonated by J. Figge, contains SV4O under the control ofthe !ac operator (see “Materials and Methods”). Identifi-cation of two transgenic founders containing these con-structs is shown in the Southern blot in Fig. 1 . Twoindependent transgenic mouse lines, both containingtandem integration of multiple copies of lad and IacO,have been established. Although transmission of inte-grated DNA sequences appears to follow Mendelianinheritance, the induction of expression of TAg is notuniform in all progeny; experiments to correlate theappropriate integration site with expression are in prog-ress. We have, however, demonstrated both inductionand reversal of TAg expression in cells from the twofounders and their descendants.

Indudion of Expression of Large I Antigen by IPTG.Fibroblast cell lines were established in the presence orabsence of IPTG from tail explants cultured from each ofthe two transgenic founders (#419 and #425), and twotransgene-negative (normal) siblings (#422 and #424).Expression of TAg was monitored by immunofluores-cence using a specific monoclonal antibody, PAB1O8(13). Two weeks after the initiation of culture in thepresence of IPTG, TAg was expressed in >80% of cells,whereas <1% of cells cultured in the absence of IPTGexpressed TAg (Fig. 2). Cells that had been cultured for27 days (since inception of the culture) without IPTG (10)

could be induced to express TAg by its addition. Ap-proximately 50% of the cells were TAg� after 2-4 days,with the intensity of fluorescence (rather than the pro-portion of TAg�) markedly increasing after 7 days. At 7days, however, these cells still stained less brightly thanthose exposed to IPTG since the initiation of culture (I”).

128 Reversible Expression of TAg in Transgenic Mice

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fig. 1. Detection of positive transgenic progeny by Southern blot hy-

hridization. icoRl-cut genomic DNA from mouse tails is shown afterblotting and probing with the plasmid containing SV4O large TAg, pSVlacO(see “Materials and Methods”). Of 70 progeny obtained from the microin-jections, two were positive, and the DNA from these founders (#419 and#425) is shown along with that from two normal littermates. The inte-grated Dcl fragment contains the SV4O early polyadenylation signal I 1 1 1.

Therefore, the pSVIacO probe detects the Dc! gene (3.4 kilobases) aswell as the SV4O fragments 14.1, 5.8, 6.6). The ratio lacI:lacO is at least10:1, which is consistent with the fact that the plasmids were injected at

a ratio of 10:1 (see ‘Materials and Methods”). The identity of the ladband has been confirmed by probing EcoRl-cut DNA with a double

purifi�cl I)r()be lacking any 5V40 sequence, as desc ribed in “Materials andMethods” (data not shown). Tandem integration of both plasmids is

indicated by restricting genomic DNA troni each founder with enzymeswhich cut only once within one of the two plasmids (e.g., Apal and BsIEII

both ( ut pRSV-1 once, hut have no sites in pSVlacO(. Hybridization to a

single lidenticall band (unique for each founder) by both probes (pRSV-

1 and pSVlacO( suggests that both fragments are integrated into theidentical site in that founder (data not shown).

Removal of IPTG from l� cells resulted in marked reduc-tion in the proportion of TAg� cells within 7 days (Fig. 3),but 1 5-30% of cells remained TAg� after 30 days.

The levels of TAg mRNA were assayed in the presenceof inducer. mRNA, isolated from fibroblasts grown in thepresence of IPTG, was detected with a SV4O-specificIacO probe (12), whereas no mRNA was detected undercomparable hybridizing conditions using mRNA fromcells cultured without IPTG, or from cultures derivedfrom nontransgenic littermates (Fig. 4A). That the pres-ence of TAg-specific mRNA is strictly dependent uponthe continued presence of inducer is illustrated in Fig.4C. The removal of IPTG results in a sharp reduction inTAg messenger by day 4, and by days 10-20, TAg mRNAcan no longer be detected. Curiously enough, lad mRNA

has not been detectable in any samples (possibly due toinstability of the prokaryotic message). However, ladexpression is implied by the influence of inducer uponexpression of TAg, fibronectin, focus formers, agarosecolonies, and cell growth.

The presence of TAg has also been detected by met-abolic labeling experiments. Exponentially growing fibro-blasts were incubated with [35S]methionine for 2-4 h,and TAg was quantitated by immunoprecipitation withmonoclonal antibody. The data given in Fig. 5 confirmthe observations that SV4O T antigen is synthesized fol-lowing the addition of IPTG and that synthesis ceasesupon removal of the inducer.

Induction of Transformation by IPTG. Initially, a qual-itative estimate was made to determine the frequency ofcell transformation in the presence of inducer. Fibroblastswere plated at saturation density, and the formation offoci was observed (14). Significantly greater numbers offoci of transformed colonies (approximately 15-fold)were formed in the presence of IPTG than in its absence.Fibroblasts from a nontransgenic sibling, #422, producedfew foci, either in the presence or absence of inducer(Fig. 6). Two quantitative assays were then used to furthercharacterize this transformation event. First, the fre-

quency of focus formation was ascertained by platingtransgenic fibroblasts at low density (100 or 500 cells/60-mm plate) on confluent monolayers of the contact-inhib-ited fibroblast line B/C-N and scoring 21 days later forfocus formation (Fig. 7). In the presence of IPTG, 78% ofthe cells formed foci, whereas no foci (<0.1%) were

formed in its absence. Second, the ability of cells to formcolonies in agarose (anchorage-independent growth) wasassayed. Ninety-two % of the cells formed agarose col-onies in the presence of IPTG, as compared to 4% in 10

controls. Quantitation of the focus-forming and agarosecolony assays is illustrated in Fig. 8. Addition of IPTG to10 cells resulted in induction of agarose colonies, but notfoci, within 7 days. Removal of IPTG resulted in thereduction of both kinds of colonies.

Fibroblasts from the transgenic mice are generally ableto grow for an extended period in the presence of IPTG(some have been cultured in IPTG for more than 300divisions), whereas fibroblasts not exposed to PIGundergo senescence (usually by 40 divisions). We have

observed the appearance of putative operator-constitu-tive (0’) mutants in cultures devoid of IPTG in whichTAg is expressed constitutively.

To obtain more precise biochemical evidence that theTAg was affecting the expression of specific genes, thesynthesis of fibronectin in tail fibroblasts was analyzed.An inverse correlation is known to exist between expres-sion of oncogenes and fibronectin (reviewed in Ref. 1 5).Therefore, cells that do not express TAg should producemore fibronectin than the corresponding cells that doexpress TAg. Fibroblasts were metabolically labeled with[35Sjmethionine, and the fibronectin that was immuno-precipitated from cell lysates was electrophoresed bysodium dodecyl sulfate-PAGE (Fig. 9). Cells grown in theabsence of IPTG (10) produced more fibronectin (2.6-fold)than those grown in its presence (I�). For comparison, B/C-N (a normal fibroblast cell line) expressed 3-fold morefibronectin than the L24 SV4O virus-transformed cell line

(described in Fig. 6).Tissue-specific Expression. Several points are note-

worthy with respect to tissue-specific expression of the

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Cell Growth & Differentiation 129

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fig. 2. Induction of SV4O TAg immunofluorescence of cultured tail fibroblasts. Tail fibroblasts grown from transgenic founders #425 and #419 andnormal sibling, #424, were stained with anti-TAg )PAB1O8) as described in ‘Materials and Methods.” Light-field photographs: (a) #425 0 and (e) #425 l�;

immunofluorescence of transgenics #425 and #419: (hI #425 I’, If) #425 I�, (C) #419 10, and (g) #419 l�; immunofluorescence of the nontransgenic

sibling: (dl #424 i#{176}and (hI #424 l�. The #425-derived cells were photographed using a Nikon microscope with 25x objective’: the remainder were’photographed with a Zeiss Axiophot microscope using a bOx objective.

transgenes in these mice. The in vivo pattern of SV4O

expression in these animals (in the absence of IPTG) is

typical ofthat described by otherworkers (1-3, 10). Brain

tumors arise in the choroid plexus in mice from bothtransgenic lines at 7-8 months. Thymic and renal hyper-

plasia have also been seen in some of these animals. Celllines established from the brain tumors stain brightly fornuclear TAg without exposure to IPTG. The onset ofexpression of TAg in bOth of our transgenic lines (asassessed by appearance of tumors) appears to be delayedwhen compared to most other transgenic lines in whichSV4O expression is controlled by the endogenous SV4O

promoter (1-3). Such a delay could be due to the effec-

tive repression of TAg expression by the lac repressor.The appearance of tumors in these animals represents anescape from immune surveillance, and quite probably

from lad control. The escape from lad control could beexplained by differences in lad expression in different

tissues under the control of the RSV promoter, the ap-pearance of O� mutations, or mutations inactivating lad.The presence of prokaryotic sequences on both injected

constructs could also affect differential expression of the

two plasmids in particular tissues. The third explanation

is unlikely, as such mutations are recessive, and lad is

present in multiple copies in the germlines of both

founders.

Any accurate assessment of the influence of the lac

repressor upon TAg expression in these animals requires

experiments involving prior induction with IPTG. Studies

are in progress to determine whether an effective levelof IPTG can be sustained systemically in vivo. In a prelim-

mary experiment with IPTG injected i.p., the only mor-

phological difference observed was a hyperplastic kidneyin one transgenic #425 progeny. Studies of tissue-specific

expression in kidney, brain, muscle, liver, and hemato-

poietic cells in vitro in the presence of inducer are in

progress.

If dominant O� mutations are the cause of tumors in

vivo and the appearance of TAg-constitutive variants in

vitro, in the absence of IPTG, then the inclusion of two

or more tandem lac operators preceding the oncogene

should reduce the frequency of expression of this mu-

tation in future transgenic animals. Constructs bearing

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fig. 3. Disappearance of SV4O nudear antigen after removal of IPTG.Fibroblasts were grown in the presence (+1or absence (-I of IPTG forvarying lengths of time prior to plating at 1 x iO� cells/well on Lab-Tek

Chamber slides. Cells were cultured on the slides for 2 days beforeimmunofluorescent staining. In all instances, at least 100 cells were

counted, and the percentage of positive staining cells was calculated. Inthis experiment, 81% of the #425 l� cells and 94% of the #419 l� cells

were positive, and no 10 cells from either founder (#425 or #419) stained.

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Fig. 4. Induction and reversal of large T antigen mRNA. A, a 2.5-kilobasemRNA for SV4O TAg is induced in the presence of IPTG (42511 andabsent when the inducer is absent (4251#{176}).No SV4O T antigen message

can be detected in mRNA from fibroblast cultures derived from a non-transgenic littermate (#422 I�). RNA from L24, an SV4O-transformed liver-

derived cell line is also shown. C, effect of removal of IPTG from #425 I�on expression of SV4O mRNA. Polyadenylated RNA (20 Mg) purified at

time zero from #425 I� and #425 10. The effect of removal of IPTG onexpression of the lacO message is seen (425l’� -e 1, days 4, 10, and 20).B and 0 the ethidium bromide-stained gels confirm the integrity of themRNA loaded in all slots.

duction. Direct correlation of data obtained from study-ing populations of cells (mRNA, TAg synthesis) with re-suIts from experiments that score single cells (focus for-mation, agar cloning, fluorescent staining) may not bepossible, because the thresholds for particular cell func-tions may differ, producing apparent discrepancies be-

tween TAg expression and the expression of a particularphenotype. At this time, the relationship of mixed pop-

ulation parameters to individually assayed cell functionsis unclear because the thresholds above which cell trans-formation can occur, or below which reversal of trans-formation can occur, with concomitant expression ofdifferentiated function, are unknown. Jat et a’l. (10) haveshown that small increases of TAg (2.5-fold) are sufficient

to maintain immortalization of fibroblasts containing the

130 Reversible Expression of TAg in Transge’nic Mice’

multiple operators have been shown to function in trans-fected mammalian cell lines (11).

Discussion

The bacterial lac operon can effectively regulate foreigngenes transfected into mammalian cells (11, 16-23). Wehave shown that the regulatory mechanism of the lacoperon can control the transformation of cells derivedfrom transgenic mice carrying SV4O TAg. Cells grown inthe presence of IPTG form foci, form colonies in agarose,synthesize nuclear TAg, and manifest reduced levels offibronectin. Upon removal of inducer, SV4O mRNA andTAg protein decrease significantly within 4-7 days; morethan 50% of cells recover their normal phenotype by day7 in that they can no longer grow as colonies in agaroseor form foci. However, even after 30 days, a significantnumber of cells remain TAg’�, and we have not yetidentified the reason for the residual TAg’� cells. It maybe that the control system of lacl/O is inefficient or that0’ mutations are selected for in some cells in the absenceof inducer, at the same time that other cells undergoreversal to the normal phenotype.

We must account for the apparent inconsistency ob-served in the shift from I� to 1 where the TAg proteinand rnRNA levels are markedly reduced in comparisonto a small reduction in the number of foci. Similarly, theshift from 1#{176}to l� can be seen clearly at the level of TAgsynthesis, but the individual assays for transformationshow marginal alterations. The retention of the trans-formed phenotype following removal of IPTG may reflectpopulation heterogeneity, which is averaged when meas-uring the TAg level. The fact that only a small proportionof cells display the transformed phenotype upon de novoaddition of IPTG may again reflect the heterogeneity inthe population. The threshold of TAg synthesis necessaryfor induction may be sufficiently high that not all cellsexhibit the cellular changes concomitant with TAg pro-

+ “ .4, �1� .FtLfl U) IL) Lt) Lt)C%J OJ C’J c%J oJ� � � �

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425I’�’

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425 I� _________________

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TAg-SpecifiCpredpftate (as % �totaI ICA predpftate)

Fig. 5. Cells which were approximately 70% confluent were labeledwith [355]methionine as described in ‘Materials and Methods.” I�-eI in

this case refers to cultures which were labeled 5 days after removal ofIPTG. l#{176}-ol�,cultures which were exposed de novo to 5 mxi IPTG 5 daysprior to labeling. Samples were precipitated using mouse anti-SV4O large

TAg (PAB1O8) as the primary antibody. The lysate was then incubated

with a secondary antibody, rabbit anti-mouse lgG. Precipitates were

washed, resuspended in radioimmunoprecipitation assay buffer (30), andcounted in Ecolume scintillation fluid.

Cell Growth & Differentiation 131

d

SV4O mutant strain tsA58 thermolabile TAg gene. Fur-thermore, in mixed populations, high levels of TAg may

be produced by only a proportion of the cells, andexperiments with cloned lines are under way to verifythe conclusions from the population studies presentedhere. Therefore, the effect ofaddition or removal of IPTGon phenotypic expression of individually scored cellfunction would not necessarily be directly proportional

to TAg expression.

fig. 6. Induction of foci of con-

tact-noninhibited cells. Repre-

sentative dishes are from an invitro assay for transformation in

which tail fibroblasts derivedfrom either transgenic #425 (a

and dl or #419 (b and e) or froma nontransgenic sibling, #422 (cand I), were monitored for theformation of foci (14) after plat-ing at saturation density in thepresence [l� (a-c)] or absence [10

(d-f)] of IPTG.

These transgenic mice are directly useful for obtaining“immortal” cell lines from any tissue in which the TAggene is expressed functionally. The strategy used in thisstudy has the potential to permit the establishment ofvirtually any cell type in culture and presents the oppor-tunity for isolating growth factors and hormones fromany tissue source. Inducible TAg expression can be tar-geted to particular tissues in transgenic mice by theinclusion of tissue-specific promoters in the microin-jected construct, as has been previously demonstratedin other systems (4-9). The lacl/O regulatory system canalso be used in conjunction with other oncogenes (e.g.,src, los, myc, jun) in order to provide a source for aparticular cell lineage for use in studying the regulationof differentiation in vitro. It seems a reasonable expecta-tion that any dividing cell could be cloned in vitro andreturned to its in vivo milieu to obtain a restoration ofnormal development. Taken to its limit, even a single cellembryo might be propagated in culture indefinitely if theappropriate oncogene were used. Embryo cells couldthen be reimplanted after removal of inducer into apseudopregnant recipient to obtain a litter of identicalprogeny.

If progress is made in controlling the in vivo levels ofIPTG, the lacl/O system might be used to regulate tran-scription units encoding toxins. This would permit theelimination in vivo of any given lineage at any given time.The effect of deleting a particular cell type on the devel-opment of the organism could then be studied.

It is evident that a relatively straightforward means fordeveloping and exploring the cell differentiation processis now feasible.

Materials and Methods

Construction of Transgenic Mice. Single cell embryosderived from superovulated ICR/Hsd female mice weremicroinjected according to standard methods for con-

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� focus-formation �1 growth in agar

fig. 8. Comparison of transformed properties of cells (focus formationand anchorage-independent growth). Data are presented as transforma-tion efficiency, which is determined by dividing either the number offocus formers or agarose colonies by the number of colonies that growin the absence of the B/C-N monolayer (see legend to Fig. 7).

132 Reversible Expression of TAg in Transgenic Mice

fig. 7. Induction of focus formers. Fibroblasts from the #425 founder were plated at low density in the presence or absence of B/C-N cells (see “Materials

and Methods”). a-d, the number of cells that grew (of the 100 plated) on plastic in the dishes lacking the B/C-N monolayer, indicating the number ofviable cells plated. e-h, focus formation on the B/C-N monolayer. 1 x 10� 10 transgenic fibroblasts were cultured in either the absence of IPTG (a and e)or 7 days after de novo addition of 2 mxi IPTG [l0��el+ lb and f); I� in the presence of 2 mea IPTG Ic and g)]; or 7 days after its removal [l�-eI (d and h)].For the 3 weeks during which foci developed, 2 mea IPTG was present only in dishes b, c, I, and g. At the same time, equivalent numbers of cells fromthe same cell suspensions were plated in agarose. In the appropriate dishes, IPTG was present at a concentration of 5 mea in both agarose layers. Agarosecolonies were counted after 3 weeks. The results of these parallel experiments are summarized in Fig. 8.

structing transgenic mice (24) with BgII-Iinearized pRSV-1 [pBR322 plasmid containing the lac repressor (1 1 )] andBamHI-cut pSVIacO [truncated pBR322 plasmid contain-ing a 22-base pair synthetic oligonucleotide encoding the/ac operator upstream of the SV4O large I antigen (12)]at ratios of 10:1, 5:1, and 1:1 copies of each sucrosegradient-purified, linearized plasmid, respectively. Bothplasmids were purified twice by CsCI density gradientsedimentation prior to linearization. The manipulatedembryos were implanted into ICR/Hsd pseudopregnantfemales, and the only founders obtained were derivedfrom the 10:1 plasmid stock. The lad-containing plasmidis regulated by the promoter from the 3’ long terminal

repeat of the Rous sarcoma virus, whereas SV4O utilizesits own endogenous promoter.

Detection of SV4O TAg and lad Genes

The probe used to detect integration or expression ofthe lad plasmid was the HindIII-NcoI fragment frompRSV-1 (1 1), whereas lacO was detected using BamHI-EcoRl-cut pSVlacO (12). Both probes were purified intwo rounds by electroelution from agarose gels. Thepresence of SV4O TAg sequences was also detected bypolymerase chain reaction amplification of ear punches,as described by Chen and Evans (25). The upstreamprimer starting at nucleotide position 51 1 7 (Genbank) is5’-TTCTAGGTCTTGAAAGGAGTGCCTGG-3’, whereasthe downstream primer found 3’ of the intron at position4483 is 5 ‘-GAGAGTCAGCAGTAGCCTCATCATCAC-3’.

Cell Culture and Staining by Immunofluorescence

Tail fragments were treated with collagenase overnightand grown in vitro in either the presence or absence of2-10 mM IPTG (BRL), in RPMI 1640 medium containing10% FCS supplemented with Mishell-Dutton cocktail(26). Prior to staining, cells were trypsinized with 0.25%trypsin-verseneand diluted to 1 x i0� cells/well on NuncLab-Tek Chamber slide (1 77402) chambers. Cells werewashed in PBS, fixed with 95% ethanol (1 mm), stainedwith 0.06% Evans blue (3 mm), washed in PBSAE, andincubated with a mouse lgG2a monoclonal antibody,PAB1O8, which reacts with a denaturation-resistant de-terminant on the NH2-terminal region of the SV4O largeand small T antigens (1 3). This antibody was provided byS. Simons (The Salk Institute). After again washing inPBSAE slides were stained with fluorescein isothio-cyanate-goat anti-mouse IgG (2 mm) and washed oncemore (30-60 mm). Coverslips were mounted with Tris-

Cell Growth & [)ifferentiation 133

1 2 3 4

_. :� � Xs3:.’�t .*- FN

LAJL4�JHfig. 9. Effect of IPTG on fibronectin (FN( synthesis. Transgenic #425

cells grown in the presence or absence of inducer were metabolicallylabele’d with [“S]methionine (ICN Translabel) using the procedures de-scribed in “Materials and Methods.” Fibronectin precipitates were’ ana-

lyzed by S”/o sodium dodecyl sulfate-PAGE with a 3.5% stacking gel. Lane

1, 10; Lane 2, l�; Lane .3, B/C-N; Lane 4, L24. The’ de’nsitometry readingswere performed on the film shown here, which was developed after a 2-h exposure using an LKB scanning densitometer.

buffered glycerol, pH 8.5. The secondary reagent is afluorescein-conjugated goat anti-mouse lgG (obtainedfrom Tago) used to reveal the presence of the large Iantigen.

Isolation of mRNA and Northern Blot Hybridization

Total RNA was prepared from the established tailfibroblast lines by the method of Chomczynski and Sac-

chi (27). mRNA was purified over an oligo-dT column.Equivalent amounts of mRNA were loaded on each lane(20 �zg). The integrity of purified RNA was confirmed byboth the presence of 28S and 185 rRNA bands as de-tected by ethidium bromide staining and by hybridizingwith a probe made from human j3-actin (28). All probesused for hybridization were labeled with [32P]dCTP (ICN)by random priming [Amersham (29)].

Metabolic Labeling of SV4O TAg or Fibronectin

Cultures in log phase (approximately 70% confluent)were labeled with [35S]methionine (ICN Translabel) inDulbecco’s modified Eagle’s medium minus methionine

for 2-4 h after 1 h of methionine starvation in this

medium. Then the cells were directly lysed in radioim-

munoprecipitation assay buffer (30) from which SV4Owas precipitated, or in cell lysis buffer [2% deoxycholate,

0.02 M Iris (pH 8.8), 2 mt�i EDTA, 2 m�i iodoacetic acid,2 m�i ethylmaleimide, and 1% Nonidet P-40], from which

fibronectin was isolated. After lysis, monoclonal PAB1O8

was added to SV4O lysates, followed by either rabbit anti-

mouse IgG or Sepharose-Protein A conjugate (RepligenIPA-300). Fibronectin was precipitated in a sandwichusing as primary antibody a rabbit anti-human fibronectin(Cappel 0211), which cross-reacts with mouse fibronec-tin, followed by a goat anti-rabbit secondary antibody.Secreted fibronectin was not assayed in these experi-ments. Cell lysates were run on 5% PAGE with a 3.5%stacking gel when fibronectin was electrophoresed.

Assay of Transformation

Quantitation of Frequency of Contact-noninhibitedCells. Induction of foci was measured by placing eitherat saturation density or at low density in the presence ofa monolayer of B/C-N cells. B/C-N is a cloned contact-inhibited line of fibroblasts derived from BALB/cKe fetal

tissue (31). B/C-N cells were plated to form a monolayerin RPMI 1640 medium supplemented with 10% fetal

bovine serum and 10% Mishell-Dutton cocktail (26) at 5x 10#{176}cells/60-mm tissue culture dish 24 h prior to theaddition of cultured tail fibroblasts. Maintenance and

staining of the cells were uniform whether cells were

plated at high or low density. Medium was changedpartially (one-half volume exchanged) once/week, and 2

weeks later, dishes were fixed with 95% ethanol (1 mm)and stained with 0.2% crystal violet (3 mm).

Quantitation of Anchorage-independent Growth.After trypsinizing and washing, harvested fibroblasts wereresuspended in RPMI 1640 medium supplemented with

25% FCS containing a final concentration of 0.35% Sea-

plaque (FMC) agarose. The top agar Containing the cellswas plated on a solidified agarose substrate consisting of

RPMI 1640 medium supplemented with 20% FCS andglutammne with Seakem agarose (FMC) at a final concen-tration of 0.5%. After 3 weeks, colonies were countedusing a New Brunswick plaque counter (Model C-i 10).

Acknowledgments

The generosity of Dr. M. C-T. Hu (11) and Dr. I. Figge (12), who provided

the two plasmids that made these transgenics possible, is gratefully

acknowledged. The authors are also grateful to Enid Ke’yser, Georgina

Sham, and Richard Dargusch for superb technical assistance. They ac-

knowledge the inspiration derived from discussions with Dr. Rod Lang-man and would like’ to thank Dr. Steve’ Baird for preparation of niurine

histological sections and advice. They thank Ron Newby for advice,

assistance, and reagents used for the fibronectin assays. The’y acknowl-

edge the help of Carol Sweeney in preparing this manuscript.

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