Proc. NatI. Acad. Sci. USA
`Vol. 77, No. 6, pp. 3567-3570, June 1980
`Genetics
`
`Transformation of mammalian cells with an amplifiable
`dominant-acting gene
`(animal cell vectors/methotrexate resistance/gene amplification)
`M. WIGLER*, M. PERUCHO*, D. KURTZ*, S. DANAt, A. PELLICERt, R. AXELt, AND S. SILVERSTEINt
`*Cold Spring Harbor Laboratories, P.O. Box 100, Cold Spring Harbor, New York 11724; and tInstituteof Cancer Research, Columbia University,
`701 West 168th Street, New York, New York 10032
`Communicated by J. D. Watson, March 10, 1980
`
`We have transferred a mutant hamster gene
`ABSTRACT
`coding for an altered dihydrofolate reductase to wild-type cul-
`tured mouse cells by using total genomic DNA from metho-
`trexate-resistant Chinese hamster ovary A29 cells as donor. By
`demonstrating the presence of hamster gene sequences in
`transformants we have provided direct evidence for gene
`transfer. Transformants selected for increased resistance to
`methotrexate contain increased amounts of the newly trans-
`ferred gene. We have used this mutant dhfr gene to introduce
`the Escherichia coli antibiotic resistance plasmid pBR322 into
`animal cells. Amplification of the dhfr sequences results in
`amplification of the pBR322 sequences as well. The use of this
`gene may allow the introduction and amplification of virtually
`any genetic element in various new cellular environments.
`The ability to transfer purified genes into cultured cells provides
`a unique opportunity to study the function and physical state
`of exogenous genes in new cellular environments. The devel-
`opment of systems for DNA transfer in animal cells originated
`with the lytic transfection of cells by using purified viral DNA
`(1, 2) and progressed to the stable transfer of viral transforming
`functions to appropriate recipient cells (3). Subsequently, viral
`genes from the herpesviruses coding for the biochemically se-
`lectable marker thymidine kinase (TK) (4-6) were transferred
`to enzyme-deficient mutant cells. Restriction fragments of
`herpes simplex virus type 1 encoding TK were isolated (6) and
`subsequently cloned into bacterial plasmids (7). Through the
`use of this selectable marker, virtually any gene can now be
`introduced into recipient cells (8, 9); however, these cells must
`be tk- mutants. Other potential selection systems are available,
`and several laboratories have recently demonstrated the
`DNA-mediated transfer of cellular genes coding for selectable
`markers such as TK (10), adenine phosphoribosyltransferase
`(11) and hypoxanthine phosphoribosyltransferase (12, 13).
`Dominant mutant cellular genes coding for drug resistance
`in principle could serve as generalized biochemical vectors for
`wild-type cells. Cultured mammalian cells are exquisitely
`sensitive to the folate antagonist methotrexant (Mtx). Mtx-
`resistant cell lines have been identified in three categories: (i)
`cells with decreased uptake of this drug (14, 15); (ii) cells that
`produce inordinately high levels of dihydrofolate reductase
`(DHFR) (16, 17); and (iii) cells with structural mutations which
`lower the affinity of DHFR for Mtx (18). When they were ex-
`amined, cells producing high levels of DHFR were found to
`contain increased copy numbers of the dhfr gene (gene am-
`plification) (19). An interesting Mtx-resistant variant cell line
`(A29) has been identified that synthesizes increased amounts
`of a mutant DHFR with decreased affinity for Mtx (18). We
`have used genomic DNA from this cell line to transfer the
`
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be hereby marked "ad-
`vertisement" in accordance with 18 U. S. C. §1734 solely to indicate
`this fact.
`
`mutant dhfr gene to wild-type Mtx-sensitive cells. Exposure
`of Mtx-resistant transformed cells to increasing levels of Mtx
`selects for cells that have amplified the transferred gene.
`
`MATERIALS AND METHODS
`Cell Culture. Mouse Ltk- aprtV cells (11) and NIH 3T3 cells
`(20) (the latter generously provided by R. A. Weinberg) were
`maintained in Dulbecco's modified Eagle's medium containing
`10% calf serum and antibiotics (growth medium). Chinese
`hamster ovary (CHO) cells and A29 cells (18), Mtx-resistant
`CHO derivatives (generously provided by L. Siminovitch), were
`maintained in growth medium supplemented with 3 times the
`usual concentration of nonessential amino acids. A29 cells were
`grown in the presence of Mtx at 20 ug/ml.
`Extraction, Restriction Endonuclease Digestion, and Li-
`gation of DNA. High molecular weight DNA was extracted
`from cultured cells as described (9). DNA was analyzed by
`electrophoresis in 0.3% agarose gels with restriction fragments
`of herpes simplex virus DNA as markers. Only DNA whose
`molecular weight average was 35 X 106 or greater was used for
`transformation experiments. CsCI/ethidium bromide-purified
`form I DNA of the Escherichia colt plasmid pBR322 was iso-
`lated from cultures of E. «ft strain HB101 (21). A29 DNA and
`pBR322 were mixed at 3:1 mass ratios, completely digested with
`restriction endonuclease Sal I (under conditions recommended
`by supplier, Bethesda Research Labs), extracted once with
`aqueous buffer-saturated phenol/chloroform/isoamyl alcohol
`25:24:1 (vol/vol), and once with chloroform/isoamyl alcohol,
`24:1, and precipitated with ethanol. DNA was resuspended and
`ligated with T4 DNA ligase (Bethesda Research Laboratories,
`Rockville, MD) at 100 ,gg of DNA and 3 units of ligase per ml
`at 40C for 24 hr in the buffers recommended by the supplier.
`The ligation product was reextracted with phenol/chloro-
`form/isoamyl alcohol and precipitated with ethanol.
`Transformation and Selection. Ltk- aprF cells and NIH
`3T3 cells were transformed with genomic DNA by the calcium
`phosphate coprecipitation method (2) as described (11). All
`DNAs were sterilized by ethanol precipitation and resuspended
`in 1 mM Tris-HCl/1 mM EDTA, pH 7.9. For tk+ transforma-
`tion, cells were exposed to hypoxanthine/aminopterin/thy-
`midine selective medium as described (10). Transformants
`resistant to Mtx were select~d in growth medium containing
`either 0.1 or 0.2 jqg of Mtx per ml with the same feeding
`schedule as for tk selection. Afte 2-3 weeks, colonies were
`isolated from individual dishes with cloning cylinders to ensure
`that each transformant arose from an independent event. In
`transformation with ligated DNAs, no more than 1 ,ug of
`pBR322 DNA was added to 106 cells per dish because higher
`
`Abbreviations: DHFR, dihydrofolate reductase; Mtx, methotrexate
`(amethopterin); TK, thymidine kinase; CHO, Chinese hamster ovary;
`kb, kilobase.
`
`3567
`
`Merck Ex. 1035, pg 1114
`
`

`

`Proc. Natl. Acad. Sci. USA 77 (1980)
`
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`Chinese hamster dhfr sequences are present in mouse
`FIG. 1.
`cells. Mouse Ltk- aprt- cells were transformed to Mtx resistance and
`their DNAs were examined for the presence of CHO sequences by
`molecular hybridization to 32P-labeled pdhfr-21 DNA. The hybrid-
`ization profiles of 20 ,ug of HindIII-cleaved DNA from dhfr-amplified
`mouse (lane M) and the dhfr-amplified CHO line A29 (lane H) are
`shown along with the HindIII patterns from four Mtx-resistant cell
`lines derived after transformation and selection at 0.1 ,ug of Mtx per
`ml (lanes A, D, G, and J). Each ofthese cell lines was also grown at Mtx
`levels of 1 ,ug/ml (lanes B, E, H, and K) and 10 pg/ml (lanes C, F, I, and
`L) and scored for amplification of dhfr sequences.
`
`intensity of unamplified dhfr. A series of additional bands were
`observed whose molecular weights were identical to those of
`restriction endonuclease-cleaved hamster dhfr gene. The 17.9-,
`7.9-, and 1.4-kb bands observed in hamster DNA are diagnostic
`for the presence of the hamster dhfr gene and were present in
`all transformants although in disproportionate intensities. In
`similar studies of NIH 3T3 Mtx-resistant transformants, 12 of
`12 contained bands diagnostic of hamster sequences (data not
`shown).
`In initial experiments, we chose the lowest concentration of
`Mtx (0.1-0.2 ,ug/ml) that would decrease survival of the mouse
`cells to <f0-7. Previous studies (18) suggested that the presence
`of a single mutant dhfr gene can render cells resistant to this
`concentration of Mtx. Comparison of the band intensities of the
`hamster dhfr gene fragments of transformed cell DNA with
`A29 suggest that our transformants contain fewer Mtx-resistant
`hamster genes than do donor A29 cells.
`Amplification of the Transferred dhfr Gene. Initial Ltk-
`aprt- transformants were selected for resistance to relatively
`low levels of Mtx (0.1 ,ug/ml). For each transformant, however,
`it was possible to select cells resistant to increased levels of Mtx
`by exposing mass cultures to successively increasing concen-
`trations of this drug. In this manner, we isolated cultures re-
`sistant to 40 ,ug of Mtx per ml, starting from clones that were
`initially resistant to 0.1 ,ug/ml. We next determined if increased
`resistance to Mtx in these transformants was associated with
`amplification of a dhfr gene and, if so, whether the endogenous
`mouse or the newly transferred hamster gene was amplified.
`DNAs from four independent transformants and their highly
`resistant derivatives were examined by blot hybridization. In
`each instance, enhanced resistance to Mtx was accompanied
`by an apparent increase in the copy number of the hamster
`gene. This is most readily seen by comparing the intensities of
`the 1.4-kb band (Fig. 1). In no instance did we detect amplifi-
`cation of the endogenous mouse dhfr gene. Lastly, different
`lines selected at equivalent Mtx concentrations appeared to
`contain different amounts of the dhfr gene.
`
`3568
`
`Genetics: Wigler et al.
`
`concentrations of pBR322 inhibited transformation. Ltk- aprt-
`DNA was added as carrier in these cases to a final DNA con-
`centration of 20,gg per dish.
`Filter Hybridization. DNA from parental and transformed
`cells was isolated, digested with restriction endonucleases,
`electrophoresed in 0.8% agarose gels, transferred to nitrocel-
`lulose filters, and hybridized as described (9). The probes for
`these experiments were 32P-labeled nick translated pBR322 or
`pdhfr-21, a cloned cDNA copy of mouse DHFR mRNA (22)
`(kindly provided by R. J. Kaufman and R. T. Schimke).
`RESULTS
`Transfer of the Mutant Hamster Dihydrofolate Reductase
`Gene to Mouse Cells. High molecular weight cellular DNA
`was prepared from wild-type Mtx-sensitive CHO cells and from
`A29 cells, and the ability of these DNA preparations to transfer
`either the dhfr gene or the tk gene to tk- mouse cells (Ltk-
`aprt-) or NIH 3T3 cells was tested (Table 1). DNAs from both
`mutant A29 and wild-type CHO cells were competent in
`transferring the tk gene to Ltk- aprtr cells. Mtx-resistant Ltk-
`aprtV colonies were observed after treatment of cells with DNA
`from A29. No colonies were observed in cells treated with
`wild-type CHO DNA. Similarly, there were 40-fold more
`Mtx-resistant 3T3 colonies after treatment of cells with A29
`DNA than after treatment with wild-type CHO DNA. These
`data suggest that treatment of Mtx-sensitive cells with A29 DNA
`resulted in the transfer and expression of a mutant dhfr gene,
`thus rendering these cells insensitive to increased levels of
`Mtx.
`In order to test this hypothesis directly, we demonstrated the
`presence of the hamster dhfr gene in DNA from transformants
`by using the filter hybridization method of Southern (23). A
`mouse dhfr cDNA clone (pdhfr-21) (22) that shares homology
`with the hamster dhfr gene was used as probe in these experi-
`ments. DNAs from A29, from transformants, and from dhfr-
`amplified mouse cells were cleaved with HindIII, electro-
`phoresed in 0.8% agarose gels, and transferred to nitrocellulose
`filters. These filters were hybridized with high-specific activity
`32P-labeled nick translated pdhfr-21 and subjected to autora-
`diography. This procedure visualizes restriction fragments of
`genomic DNA homologous to the dhfr probe. Prominent bands
`were observed at 15, 3.5, and 3 kilobases (kb) for dhfr-amplified
`mouse DNA and at 17, 7.9, 3.7, and 1.4 kb for dhfr-amplified
`hamster DNA (Fig. 1). The restriction profiles of these two
`species were sufficiently different to permit us to detect the
`hamster gene in the presence of an endogeneous mouse
`gene.
`Four Ltk- aprt- cell transformants resistant to Mtx were
`examined in this way (Fig. 1). In each transformed cell line, we
`observed the expected profile of bands resulting from cleavage
`of the endogenous mouse dhfr gene although at the decreased
`
`DNA source
`(CHO cells)
`A29*
`
`Wild type
`
`Transformation data
`Table 1.
`Mtx-resistant
`colonies, no./total
`no. dishes
`56/5t
`161/5*
`0/5t
`4/5$
`Twenty micrograms of DNA was used to transform 106 cells per
`dish. Mtx concentration was 0.2 gg/ml.
`* CHO Pro mtxRIII (18).
`t Ltk- aprt- cells were used as recipients.
`t NIH 3T3 cells were used as recipients.
`
`tk+ colonies,
`no./total no. dishes
`25/5t
`
`30/5t
`
`Merck Ex. 1035, pg 1115
`
`

`

`Genetics: Wigler et al.
`A\
`
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`D)
`
`A
`
`Proc. Natl. Acad. Sci. USA 77 (1980)
`D)
`G
`C
`13
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`V
`
`F
`
`3569
`
`m04
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`
`FIG. 2.
`Presence and amplification of pBR322 sequences in cells
`transformed with A29-pBR322 ligates. Cells were transformed with
`the ligation product of Sal I-cleaved A29 and pBR322 DNAs.
`Transformants were selected initially for resistance to 0.1 ,gg of Mtx
`per ml. After cloning, cultures were exposed to increasing concen-
`trations of Mtx, and DNA was extracted, cleaved with Xba I, and
`analyzed for the presence of pBR322 sequences by filter hybridization.
`Lanes A-D, 10 Ag of DNA from the SS-6 line grown in 0.1, 2, 10, or 40
`;g of Mtx per ml, respectively. Lane E, 50 pg of Pst I-cleaved pBR322
`DNA.
`
`The dhfr Gene as a Generalized Transformation Vector.
`Selectable genes can be used as vectors for the introduction of
`other genetic elements into cultured cells. In previous studies,
`we have demonstrated that cells transformed with the tk gene
`are likely to incorporate other unlinked genes (9). The gener-
`ality of this approach was tested for the selectable marker, the
`mutant dhfr gene. Total cellular DNA (20 Mg) from A29 was
`mixed with 1 jig of HindIll-linearized pBR322 DNA. Recipient
`cells (Ltk- aprt-) were exposed to this DNA mixture and, after
`2 weeks, Mtx-resistant colonies were picked. Genomic DNA
`from transformants was isolated, cleaved with HindIll, and
`analyzed for the presence of pBR322 sequences. Two inde-
`pendent transformants were examined in this way, and multiple
`copies of pBR322 sequences were present in both cases (data
`not shown).
`An alternate approach to generalized transformation involves
`ligation of a nonselectable DNA sequence to a selectable gene.
`Because the mutant dhfr gene is a dominant-acting gene con-
`ferring drug resistance, it can be used as a vector. Furthermore,
`it may be possible to amplify any genetic element ligated to this
`vector by selecting cells resistant to increased levels of Mtx. To
`explore this possibility, restriction endonucleases that do not
`destroy the dhfr gene of A29 were identified by transformation
`assay. One such restriction endonuclease, Sal I, does not destroy
`the transformation potential of A29 DNA. Sal I-cleaved A29
`DNA was therefore ligated to Sal I-linearized pBR322. This
`ligation product was subsequently used in transformation ex-
`periments with Ltk- aprt- cells. Mtx-resistant colonies were
`
`As in Fig. 2. Lanes A, B, and C, 10,ug of DNA from cell
`FIG. 3.
`line SS-1 grown in 0.1, 2, and 40 Mg of Mtx per ml, respectively. Lanes
`D-F, 0lg of DNA from the clone HH-1 grown in 0.1, 2, and 40gg of
`Mtx per ml. Lane G, 50 pg of Pst I-cleaved pBR322 DNA.
`picked and grown into mass culture in the presence of 0.1 ig
`of Mtx per ml. Mass cultures were subsequently exposed to in-
`creasing concentrations of Mtx.
`DNAs were obtained from mass cultures resistant to 0.1, 2,
`10, and 40kg of Mtx per ml and the copy number of pBR322
`and dhfr sequences was determined by blot hybridization. Six
`independent transformed lines were examined in this fashion.
`Five of these lines exhibited multiple bands homologous to
`pBR322 sequences. In four of these transformed clones, at least
`one of the pBR322-specific bands increased in intensity upon
`amplification of dhfr (Figs. 2 and 3). All pBR322 bands present
`in transformant SS-6 at 0.1 Mg/ml continued to increase in in-
`tensity as cells were selected first at 2 ,g/ml and then at 40
`Mig/ml (Fig. 2). We estimate that there was at least a 50-fold
`increase in copy number for pBR322 sequences in this cell line.
`In SS-I (Fig. 3, lanes A, B, and C), two pBR322-specific bands
`were observed in DNA from cells resistant to 0.1 Mug of Mtx per
`ml. These bands increased severalfold in intensity in cells re-
`sistant to 2 ,g/ml. No further increase in intensity was observed,
`however, in cells selected for resistance to 40 ,g/ml. In a third
`cell line, HH-1 (Fig. 3, lanes D, E, and F), two pBR322-specific
`bands increased in intensity upon amplification, whereas others
`remained constant or decreased in intensity. Thus, the pattern
`of amplification of pBR322 sequences we observed in these cells
`was quite varied. Nevertheless, the mutant dhfr gene can be
`used to introduce and subsequently amplify unselected DNA
`sequences in cultured animal cells.
`DISCUSSION
`The potential usefulness of DNA-mediated transformation in
`the study of eukaryotic gene expression depends to a large ex-
`tent on its generality. Cellular genes coding for selectable bio-
`chemical functions have previously been introduced into mu-
`tant cultured cells (10-13). In the present study, we have
`
`Merck Ex. 1035, pg 1116
`
`

`

`3570
`
`Genetics: Wigler et al.
`transferred a dominant-acting Mtx-resistant dhfr gene to
`wild-type cultured cells. In initial experiments, DNA from A29
`cells, a Mtx-resistant CHO derivative synthesizing a mutant
`dhfr, was added to cultures of mouse Ltk- aprt- cells or NIH
`3T3 cells. Mtx-resistant colonies appeared at a frequency of
`about 10 colonies per 5 X 105 cells per 20 ,gg of cellular DNA.
`Upon transformation, fewer colonies were observed with NIH
`3T3 and none with Ltk- cells when DNA obtained from
`wild-type Mtx-sensitive cells was used, although this DNA was
`a competent donor of the tk gene. The Mtx-resistant NIH 3T3
`colonies obtained by using wild-type DNA as donor were not
`significantly above the spontaneous level of resistance (data not
`shown), and these colonies were not studied further. Definitive
`evidence that we effected transfer of a mutant hamster dhfr
`gene was the presence of the hamster gene in mouse transfor-
`mants in blot hybridization experiments. In all transformants
`examined, we observe two sets of restriction fragments ho-
`mologous to a mouse dhfr cDNA clone: a series of bands char-
`acteristic of the endogenous mouse gene and a second series
`characteristic of the donor hamster gene.
`The number of copies of dhfr we observed in our initial
`transformants is low. This observation is consistent with previous
`studies suggesting that a single mutant dhfr gene is capable of
`rendering cells Mtx-resistant under our selective criterion (0.1
`,qg of Mtx per ml) (18). Exposure of these initial Mtx-resistant
`transformants to stepwise increases in drug concentration results
`in the selection of cells with enhanced Mtx resistance. Blot
`analysis indicates that these cells have increased amounts of the
`newly transferred mutant hamster dhfr gene. In no transfor-
`mants have we observed amplification of the endogenous mouse
`gene in response to selective pressure. It is likely that a single
`mutant gene affords significantly greater resistance to a given
`concentration of Mtx than does a single wild-type gene. If the
`frequency of amplification is low, we are merely selecting re-
`sistant variants arising from the minimal number of amplifi-
`cation events. It is also possible that newly transferred genes
`undergo amplification more readily than do endogeneous
`genes.
`We have explored the use of the mutant dhfr gene as a vector
`for the introduction and amplification of nonselectable genetic
`elements into cultured cells. Genomic DNA from A29 cells was
`cleaved with restriction enzymes and ligated to restriction en-
`donuclease-cleaved pBR322 sequences prior to transformation.
`Most resulting transformants contained multiple pBR322 se-
`quences. In many cases, amplification of dhfr genes resulted
`in the concomitant amplification of pBR322. The patterns of
`amplification differed among cell lines. In one transformant,
`all pBR322 sequences amplified with increasing Mtx concen-
`trations. In other transformants, only a subset of the sequences
`amplified. In yet other lines, sequences appeared to be lost or
`rearranged. In some lines, amplification proceeded apace with
`increasing Mtx concentrations up to 40 gg/ml whereas, in
`others, amplification ceased at 2 ,ug/ml. It appears that the
`mechanisms of amplification may be quite varied. Whatever
`mechanisms are responsible for these complex events, it is clear
`that the dhfr amplification unit extends beyond the limits of
`the dhfr gene itself and this can be exploited to control the
`dosage of any gene introduced into cultured cells.
`
`Proc. Natl. Acad. Sci. USA 77 (1980)
`
`2.
`
`3.
`
`4.
`
`5.
`6.
`
`7.
`
`8.
`
`9.
`
`Although we have succeeded in transferring Mtx resistance
`to Ltk- aprt- cells and mouse NIH 3T3 cells, we have not as yet
`had success with various other cell lines. In these instances either
`the cells were poor recipients for DNA-mediated transforma-
`tion or they were already substantially resistant to Mtx. We
`expect that cloning a Mtx-resistant dhfr gene will overcome
`these difficulties and extend the use of this gene as amplifiable
`vector for wild-type cells.
`We thank Drs. Sol Spiegelman and James Watson for their generous
`support and C. Fraser and C. Lama for their excellent technical assis-
`tance. This work was supported by grants from the National Institutes
`of Health to R.A. and S.S. (CA-16346, CA-23767, CA-17477) and a
`Cancer Center Grant to James Watson (CA-13106). S.S. was the re-
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`10.
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`11.
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`12.
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`14.
`15.
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`16.
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`18.
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`Merck Ex. 1035, pg 1117
`
`