Proc. Natl. Acad. Sci. USA
`Vol. 76, No. 3, pp. 1373-1376, March 1979
`Genetics
`
`DNA-mediated transfer of the adenine phosphoribosyltransferase
`locus into mammalian cells
`(biochemical transformation/unique genes/mutant cells)
`MICHAEL WIGLER*t, ANGEL PELLICER*, SAUL SILVERSTEINt§, RICHARD AXEL*¶, GAIL URLAUB11,
`AND LAWRENCE CHASINII
`*Institute of Cancer Research, tDepartment of Microbiology, and IDepartments of Pathology and Biochemistry, Columbia University, 701 West 168th Street,
`New York, New York 10032; and I'Department of BiologicalSciences, Columbia University, New York, New York 10027
`Communicated by Sol Spiegelman, December 14, 1978
`
`In this report, we demonstrate the feasibility
`ABSTRACT
`of transforming mouse cells deficient in adenine phosphori-
`bosyltransferase (aprt; AMP:pyrophosphate phosphoribosyl-
`transferase, EC 2.4.2.7) to the aprt+ phenotype by means of
`DNA-mediated gene transfer. Transformation was effected by
`using unfractionated high molecular weight genomic DNA from
`Chinese hamster, human, and mouse cells and restriction en-
`donuclease-digested DNA from rabbit liver. The transformation
`frequency observed was between 1 and 10 colonies per 106 cells
`per 20 ,gg of donor DNA. Transformants displayed enzymatic
`activity that was donor derived as demonstrated by isoelectric
`focusing of cytoplasmic extracts. These transformants fall into
`two classes: those that are phenotypically stable when grown
`in the absence of selective pressure and those that are phe-
`notypically unstable under the same conditions.
`
`The DNA-mediated transfer of cellular genes, discovered (1)
`and previously exploited in the prokaryotes, has recently been
`extended to eukaryotes. Early studies on the transformation**
`of eukaryotic cells were restricted to viral genes (2, 3). Recently,
`transformation of yeast spheroplasts with recombinant DNA
`molecules has permitted the isolation and characterization of
`genes coding for selectable biochemical markers (4). In our
`laboratories we have transferred cellular genes from complex
`vertebrate genomes to cultured mammalian cells. In initial
`studies, we demonstrated transfer of the thymidine kinase (tk)
`gene of herpes simplex virus to mutant mouse Ltk- cells (5).
`After optimizing the conditions for transformation in this model
`system, we were able to transfer the cellular tk gene by using
`unfractionated high molecular weight genomic DNA from
`various species as donors (6). The transformed cell expressed
`tk activity encoded by donor DNA.
`The potential usefulness of this observation depends to a large
`extent on its generality. In principle, transformation should be
`detectable for all genes for which selection conditions are
`available. In this study, we demonstrate the transfer of the gene
`coding for adenine phosphoribosyltransferase (aprt; AMP:
`pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) to mu-
`tant cells lacking this enzyme (Ltk- aprt-). Transformants
`express aprt activity with the characteristics of the organism
`from which the transforming DNA was derived. Taking these
`results together with our previous results, we have demonstrated
`that DNA-mediated gene transfer in animal cells can provide
`a bioassay for dominant-acting genes present at concentrations
`of one part per haploid genome.
`
`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.
`
`MATERIALS AND METHODS
`Cell Culture. Murine Ltk- aprr cells are derivatives of Ltk-
`clone 1D cells (7) and were originally isolated and characterized
`by R. Hughes and P. Plagemann. They were generously pro-
`vided by R. Hughes. Cells were maintained in growth medium
`[Dulbecco's modified Eagle's mediumn containing 10% calf
`serum (Flow Laboratories, Rockville,'MD)] supplemented with
`diaminopurine at 50 ,g/ml. Prior to transformation, cells were
`washed and grown for three generations in the absence of di-
`aminopurine. HEp-2 (human), HeLa (human), CHO (Chinese
`hamster ovary), and Ltk- cells were grown in growth medium.
`For CHO, medium was supplemented with 3X the usual con-
`centration of nonessential amino acids. LH2b, a derivative of
`Ltk- transformed with herpes simplex virus tk DNA, was
`maintained in growth medium containing hypoxanthine at 15
`ttg/ml, aminopterin at 0.2 ,gg/ml, and thymidine at 5.0 jg/ml
`(HAT) (5). All culture dishes were Nunclon (Vangard Inter-
`national, Neptune, NJ) plastic.
`Extraction and Restriction Endonuclease Cleavage of
`Genomic DNA. High molecular weight DNA was obtained
`from cultured cells (CHO, LH2b, and HeLa) or from frozen
`rabbit livers as previously described (6). High molecular weight
`salmon sperm DNA was obtained from Worthington. Restric-
`tion endonucleases were obtained from New England BioLabs.
`Restriction endonuclease cleavage (Bam I, HindIII, Kpn I, and
`Xba I) was performed in buffer containing 50 mM NaCl, 10
`mM Tris-HCl, 5 mM MgCl2, 7 mM mercaptoethanol, and bo-
`vine serum albumin at 100 ,g/ml (pH 7.9). The enzyme-to-
`DNA ratio was at least two units/,gg of DNA, and reaction
`mixtures were incubated at 370C for at least 2 hr (one unit is
`the amount of enzyme that digests 1 ,ug of DNA in 1 hr). To
`monitor the completeness of digestion, 1 pl of nick-translated
`adenovirus-2 [32P]DNA was incubated with 5 pil of reaction
`volume for at least 2 hr, cleavage products were separated by
`electrophoresis in 1% agarose gels, and digestion was monitored
`by exposing the dried gel to Cronex 2DC x-ray film.
`Transformation and Selection. The transformation protocol
`was as described (8) with the following modifications. One day
`prior to transformation, cells were seeded at 0.7 X 106 cells per
`Abbreviations: tk, thymidine kinase; aprt, adenine phosphoribosyl-
`transferase; HAT, hypoxanthine/aminopterin/thymidine.
`t Present address: Cold Spring Harbor Laboratory, Cold Spring Harbor,
`NY 11724.
`§ To whom correspondence should be addressed.
`** We define transformation as a change in the genotype of a recipient
`cell mediated by the introduction of purified DNA. Transformation
`can frequently be detected by the stable and heritable change in
`the phenotype of the recipient cell that results from an alteration
`in either the biochemical or the morphological properties of the
`recipient.
`
`1373
`
`Merck Ex. 1031, pg 1075
`
`

`
`1374
`
`Genetics: Wigler et al.
`dish. The medium was changed 4 hr prior to transformation.
`Sterile, ethanol-precipitated high molecular weight or restric-
`tion endonuclease-cleaved eukaryotic DNA dissolved in 1 mM
`Tris (pH 7.9)/0.1 mM EDTA was used to prepare DNA/CaCI2,
`which contains DNA at 40 ,g/ml and 250 mM CaCl2 (Mal-
`linkrodt). Twice-concentrated Hepes-buffered saline (2X HBS)
`was prepared; it contains 280 mM NaCI, 50 mM Hepes, and 1.5
`mM sodium phosphate, pH-adjusted to 7.10 + 0.05. DNA/
`CaCd2 solution was added dropwise to an equal volume of sterile
`2X HBS. A 1-ml sterile plastic pipette with a cotton plug was
`inserted into the mixing tube containing 2X HBS, and bubbles
`were introduced by blowing while the DNA was being added.
`The calcium phosphate/DNA precipitate was allowed to form
`without agitation for 30-45 min at room temperature. The
`precipitate was then mixed by gentle pipetting with a plastic
`pipette, and 1 ml of precipitate was added per plate, directly
`to the 10 ml of growth.medium that covered the recipient cells.
`After 4-hr incubation at 37°C, the medium was replaced and
`the cells were allowed to incubate for an additional 20 hr. At
`that time, selective pressure was applied. For tk+ selection,
`medium was changed to growth medium containing HAT. For
`aprt+ selection, cells were trypsinized and replated at lower
`density (about 0.5 X 106 cells per 10-cm dish) in medium con-
`taining 0.05 mM azaserine and 0.1 mM adenine. For both tk+
`and aprt+ selection, selective media were changed the next day,
`2 days after that, and subsequently every 3 days for 2-3 weeks
`while transformant clones arose. Colonies were picked by using
`cloning cylinders and the remainder of the colonies were scored
`after formaldehyde fixation and staining with Giemsa. For
`characterization, clones were grown into mass culture under
`continued selective pressure. A record was kept of the apparent
`number of cell doublings for each clone isolated.
`Enzyme Assays. Extracts were prepared by resuspending
`washed cell pellets (approximately i07 cells) in 0.1 ml of 0.02
`M potassium phosphate, pH 7, containing 0.5% Triton X-100.
`The supernatant (cytoplasm) obtained after 25 min of 700 X
`g centrifugation was used for the quantitation of enzymatic
`activity and for electrophoresis. aprt and protein were assayed
`as previously described (s). Inclusion of 3 mM thymidine tri-
`phosphate, an inhibitor of 5'-nucleotidase (10), in the reaction
`mixture did not increase AMP recovery, indicating that the
`nucleotidase was not interfering with the measurement of aprt
`activity. Isoelectric focusing of aprt was carried out essentially
`as described for hypoxanthine phosphoribosyltransferase (11)
`with the following exceptions: The polyacrylamide gel con-
`tained an Ampholine (LKB) mixture of 0.8% pH 2.5-4, 0.8%
`pH 4-6, and 0.4% pH 5-7. For assaying enzymatic activity,
`[2-3H]adenine [0.04 mM, 1 Ci/mmol, New England Nuclear
`(1 Ci = 3.7 X 1010 becquerels)] was substituted for hypoxan-
`thine.
`
`RESULTS
`Transformation to the aprt+ Phenotype. Biochemical
`transformation occurs with low frequency and is usually de-
`tected by the ability of the rare transformed cell to grow under
`appropriate selective conditions. The development of a trans-
`formation system therefore requires a recipient cell that is both
`competent for transformation and sensitive to selection. In
`addition, the frequency at which the recipient cell sponta-
`neously reverts to selection resistance must be lower than the
`frequency of transformation. Previous results indicated that
`Ltk- cells (deficient for tk) are competent recipients for cellular
`DNA and undergo transformation to the tk+ phenotype at a rate
`of 1-10 colonies per 106 cells per 20 ,ug of donor DNA (6). The
`aprt- variant of Ltk- cells (Ltk- aprt-) grows in the presence
`of diaminopurine. aprt+ cells are selected in media containing
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`Table 1.
`
`Gene transfer with total genomic DNA
`from various species
`
`Total
`Total
`aprt+
`tk+
`colonies/ Aver- colonies/ Aver-
`age/
`age/
`total
`total
`plate
`plates
`plate
`plates
`Donor DNA
`22/5
`20/14
`Chinese hamster (CHO cells)
`1.4
`4.4
`42/4
`95/14
`Human (HeLa cells)
`6.8
`10.5
`Mouse (LH2b cells)
`100/5
`24/15
`1.6
`20.0
`0/5
`0/15
`Salmon (testes)
`0.0
`0.0
`0/5
`0/15
`None
`0.0
`0.0
`High molecular weight DNA was prepared and coprecipitated with
`calcium phosphate; 20 ,g of precipitated DNA (in 1 ml) was added
`to each plate. Transformants were scored for either the tk+ or aprt+
`phenotype after selection.
`
`azaserine and adenine. Azaserine blocks de novo purine bio-
`synthesis, and adenine can be utilized for the synthesis of purine
`nucleotides only by aprt+ cells. Ltk- aprt- cells show a low rate
`of spontaneous reversion to the aprt+ phenotype as judged by
`their cloning efficiency in azaserine/adenine (unpublished
`studies).
`Ltk- aprt- cells are therefore appropriate recipients for the
`transfer of the aprt gene. High molecular weight DNA was
`prepared from human, hamster, and wild-type mouse cultured
`cells. A calcium phosphate/DNA coprecipitate was added to
`Ltk- aprv cells under a modification of the transformation
`conditions described by Graham and van der Eb (8). After 24
`hr, cells were exposed to the selection media.
`Colonies were scored after 2-3 weeks. The results of one
`experiment are shown in Table 1. The data demonstrate that
`transformation to the tk+ and aprt+ phenotypes can be effected
`with DNA preparations from hamster, human, and mouse. The
`frequency of transformation in each case was about 10 colonies
`per 106 cells per 20 ,ug of DNA. No transformants resulted from
`treatment with salmon DNA and no aprt+ colonies arose in
`untreated cultures. This is in accord with the observation that
`the reversion of this line to aprt+ occurs with a frequency of 3
`X 10o- under these conditions (unpublished studies).
`Individual transformant colonies were picked, in cloning
`cylinders, from separate plates to ensure that they represented
`independent transformation events. These colonies were grown
`
`Table 2.
`
`aprt activities of parental and transformant clones
`aprt activity,
`nmol AMP/min
`per mg
`
`Donor DNA
`
`2.19
`
`6.52
`2.09
`
`<0.005
`
`Cell line
`Donors
`HEp-2 (human)
`CHO (Chinese
`hamster)
`Ltk- (mouse)
`Recipient
`Ltk- aprt-
`Transformants
`Mouse
`MA-1
`1.94
`MA-4
`Mouse
`2.29
`1.72
`Human
`HA-1
`Human
`1.37
`HA-4
`Chinese hamster
`0.86
`CA-3
`1.62
`Rabbit
`RA-1*
`* This clone is a mouse cell revertant isolated after presumptive
`transformation with rabbit liver DNA.
`
`Merck Ex. 1031, pg 1076
`
`

`
`Genetics: Wigler et al.
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`1375
`
`into mass cultures under selective conditions (azaserine/ade-
`nine). Cytoplasmic extracts were prepared from individual
`clones and assayed for aprt activity. As shown in Table 2, Ltk-
`aprt- cytoplasmic extracts had negligible activity. In contrast,
`all transformants displayed enzymatic activities in the range
`of wild-type mouse Ltk- cells.
`Electrophoretic Characterization of aprt. Tables I and 2
`indicate that the appearance of aprt+ colonies was dependent
`upon the addition of mammalian DNA, suggesting that gene
`transfer, rather than reversion, had occurred. Direct evidence
`for gene transfer was obtained by the electrophoretic charac-
`terization of the aprt in transformed clones. Isoelectric focusing
`in polyacrylamide gels clearly separates murine aprt from that
`of human, rabbit, and Chinese hamster (Fig. 1). Transformed
`clones derived from treatment with human or Chinese hamster
`DNA express aprts with isoelectric points characteristic of the
`donor DNA species. No murine aprt activity is detected in these
`cells. In contrast, transformants derived after treatment with
`murine DNA express the murine aprt activity. These results
`argue strongly that the selected clones do not represent a special
`class of revertants that reexpress the parental murine aprt rather
`than the donor DNA aprt. Reversion of the parental Ltk- aprt-
`clone can occur, however, as indicated in one experiment in
`which rabbit DNA was used as donor. In this case, the aprt+
`clone subsequently isolated exhibited murine aprt activity.
`Stability of the Transformed Phenotype. We next asked if
`expression of aprt was stable in the absence of selective pressure.
`Individual transformant clones, grown into mass culture under
`
`Table 4. Gene transfer with restriction endonuclease-cleaved DNA
`Total aprt+
`colonies/
`total plates
`Endonuclease
`1/9
`BamI
`0.1
`104/10
`HindIII
`10.4
`109/8
`13.6
`Kpn I
`High molecular weight rabbit liver DNA was prepared and cleaved
`to completion with the indicated restriction endonucleases. Cleaved
`DNA was used as donor in transformation experiments.
`
`Average/
`plate
`
`selective pressure, were subcultured for various times in the
`absence of selective pressure. The fraction of cells that retained
`the aprt+ phenotype was determined by measuring cloning
`efficiencies in selective and nonselective media. The results of
`these experiments (Table 3) demonstrate that transformants
`fall into two categories: stable transformants that retain the
`ability to grow in azaserine/adenine when cultured in the ab-
`sence of selective pressure (HA-1, HA-4, CA-3); and unstable
`transformants that do not (CA-1, MA-1, MA-4). The rate of loss
`of the aprt phenotype can be calculated from these data if one
`assumes that rate of loss is constant in each generation (see
`footnote §, Table 3). For the parental Ltk- (aprt+) cells, a rev-
`ertant of Ltk- aprt- (RA-1), and the stable transformants, the
`rate of loss was no more than 2% per generation. For the un-
`stable transformants, the calculated rate of loss was as high as
`27% per generation (CA-3).
`
`Cell
`line*
`Ltk-
`
`CA-1
`
`CA-3
`
`HA-1
`
`HA-4
`
`MA-1
`
`MA-4
`
`RA-1
`
`Rate of
`loss of
`aprt+
`phenotype
`per
`generations
`
`<0.01
`
`0.27
`
`0.02
`
`<0.01
`
`<0.01
`
`0.14
`
`0.15
`
`Table 3.
`
`Generations int
`Selective
`Neutral
`medium
`medium
`
`Exp.
`
`Stability of the transformed phenotype
`Relative
`cloning
`efficiency
`in
`selective
`mediumt
`23
`3
`0.89
`1
`26
`0.93
`0
`2
`0.52
`2
`37
`1
`16
`0.006
`24
`2
`3
`0.96
`42
`1
`20
`0.72
`25
`2
`54
`3
`1.00
`1
`47
`20
`0.84
`2
`3
`0.64
`41
`1
`25
`20
`0.76
`2
`39
`4
`0.28
`1
`26
`0.01
`24
`2
`38
`4
`0.23
`1
`23
`0.01
`24
`2
`3
`0.88
`47
`1
`<0.01
`20
`0.96
`31
`2
`* Ltk- is the parental line for Ltk- aprtm4 RA-1 is a revertant aprt+ derivative of the latter. All other
`lines represent aprt+ transformants of Ltk- aprt-. For derivatives and enzyme characterization see
`Table 1 and Fig. 1.
`t Clones were picked and grown in selective medium for a known number of generations. Cells were then
`grown in neutral medium for a known number of generations prior to measuring their cloning ef-
`ficiencies under selective and nonselective conditions.
`One hundred cells were plated in triplicate into selective (azaserine/adenine) and nonselective media.
`The relative cloning efficiency in selective medium is defined as the ratio of the cloning efficiency under
`selective conditions to the cloning efficiency under nonselective conditions. The latter was generally
`50-70%.
`§ In these calculations we have assumed that for any given cell line the rate of loss of the aprt phenotype
`is constant in each generation. With that assumption, the rate of loss per generation may be calculated
`by using the formula FM(1 - X)NM = FN, in which FM is the relative cloning efficiency in selective
`medium after M generations in nonselective medium; FN is similarly defined; and X is the rate of loss
`per generation.
`
`Merck Ex. 1031, pg 1077
`
`

`
`1376
`
`Genetics: Wigler et al.
`A B C D E
`F
`G H
`
`J
`
`K L M
`
`NO
`
`I
`
`I
`
`*.e
`
`a
`
`Isoelectric focusing of aprt. The high-speed supernatants
`FIG. 1.
`from homogenates of wild-type and mutant cells, transformed cells,
`and rabbit liver were focused on 4.5% acrylamide gels containing an
`Ampholine mixture of 0.8% pH 2.5-4/0.8% pH 4-6/0.4% pH 5-7. For
`development of enzyme activity, [2-3H]adenine was used and the
`product was blotted onto polyethyleneimnine-cellulose and localized
`by fluorography. A, Ltk- cell extract; B, rabbit liver homogenate; C,
`HEp-2 cell extract; D, CHO cell extract; E, extract of Ltk- aprt-
`cells transformed with HeLa cell DNA (HA-1); F, extract from cells
`transformed with CHO cell DNA (CA-3); G, extract from cells
`transformed with LH2b cell DNA (MA-1); H, extract from
`cells transformed with HeLa cell DNA (HA-4); I, extract from cells
`transformed with CHO cell DNA (CA-1); J, extract from cells trans-
`formed with LH2b cell DNA (MA-4); K, extract from an Ltk- aprt-
`revertant (RA-1); L, extract from HEp-2 cells; M, extract from CHO
`cells; N, extract from Ltk- cells, 0, extract from rabbit liver homog-
`enate.
`
`Transformation with Restriction Endonuclease-Cleaved
`DNA. It was of interest to determine whether transformation
`of aprt- cells could be performed with restriction enzyme
`fragments of DNA. This requires the use of restriction en-
`donucleases that do not cleave the aprt gene. High molecular
`weight DNA was therefore prepared from rabbit liver and di-
`gested to completion with a variety of restriction enzymes. This
`restriction endonuclease-cleaved DNA was used in transfor-
`mation assays. The data are summarized in Table 4. Although
`Bam I destroyed the ability of DNA to transfer aprt, cleavage
`of rabbit DNA with either Kpn I or HindIII did not result in
`a reduction in transformation efficiency compared with the
`efficiency obtained when the transformation was performed
`with uncleaved DNA. Subsequent experiments indicated that
`cleavage of rabbit DNA with Xba I also does not destroy the
`aprt gene (data not presented).
`DISCUSSION
`In this study we have demonstrated the transformation of aprt-
`mouse cells to aprt+, using both high molecular weight and
`restriction endonuclease-cleaved DNA from a variety of species
`as donors. Stable transformation appears at a frequency 100fold
`higher than the spontaneous reversion frequency displayed by
`this cell line. The frequency of transformation ranges from 1
`to 10 colonies per 106 cells per 20 ,ug of donor DNA. These re-
`sults together with comparisons of the aprt activities from do-
`nors and transformants by isoelectric focusing directly dem-
`onstrate that the aprt in transformants is derived from the donor
`
`Proc. Natl. Acad. Sci. USA 76 (1979)
`
`DNA and is not due to reversion or reactivation of the murine
`gene.
`The transformants we have isolated fall into two categories:
`those that retain the ability to express aprt even in the absence
`of selective pressure and those that do not. In this respect, bio-
`chemical transformants obtained after DNA-mediated gene
`transfer resemble transformants obtained after chromosome-
`mediated transfer (12, 13). The molecular basis for the observed
`phenotypic instability is not known at the present time.
`The method employed to transfer both the tk and aprt genes
`can in principle be applied to any gene for which appropriate
`selective conditions and recipient cells exist. We have, for ex-
`ample, recently succeeded in transferring the gene coding for
`a methotrexate-resistant folate reductase gene (14) to wild-type
`cells (unpublished results). The generality of these observations
`indicates that transformation will facilitate the dissection of
`complex cellular phenotypes in eukaryotic cells.
`To date, the isolation of genes from animal cells has been
`confined to those loci for which hybridization probes are
`available. Numerous interesting loci, however, are not tran-
`scribed in amounts sufficient to generate hybridization probes.
`DNA-mediated gene transfer provides a unique bioassay for
`gene function and a method that could be employed in alter-
`native approaches for gene isolation.
`We thank Dr. Sol Spiegelman for helpful discussions and suggestions.
`This research was supported by grants from the National Institutes of
`Health to R.A. (CA-18346), to L.C. (GM-22629), and to S.S. (CA-
`17477). M.W. was supported by a Postdoctoral Fellowship from the
`National Institutes of Health (GM-06877). S.S. was the recipient of a
`Research Career Development Award from the National Institutes of
`Health (CA-0049).
`
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`Merck Ex. 1031, pg 1078