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`VIOLECULAR
`AND APPLIED \.
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`
`/OLUMF 1-NIJMBEP A-1989-RAVEN PPESS
`
`Genzyme Ex. 1004, pg 57
`
`

`
`Journal of
`Molecular and Applied Genetics
`
`Editor-in-Chief:
`
`Howard M. Goodman, Ph.D.
`
`Department of Molecular Biology
`Massachusetts General Hospital
`Boston. Massachusetts 021 I4, U.S.A.
`
`Editorial Board
`
`John Abelson, La Jolla
`Frederick Ausubel, Catnbritlge
`John R. Bedbrook, Canberra
`Lawrence Bogorad, Cambridge
`Mary-Dell Chilton, St. Louis
`Richard A. Flavell, London
`Raymond F. Gesteland, Salt Lake City
`Gary Gussin, Iowa City
`Dean H. Hamer, Bethesda
`Masayori lnouye, Stony Brook
`Robert Kamen, London
`Yuet Wai Kan. San FI‘tlIl('i.i‘(‘t)
`Laurence H. Kedes, Palo Alto
`Daniel Klessig, Salt Lake Cit)‘
`Thomas Maniatis, Cambridge
`David W. Martin, Jr., San Francisco
`Marc Van Montagu, Gent
`Richard C. Mulligan, Cambridge
`Alexander Rich, Cambridge
`Jet!‘ Schell, Cologne
`James Shepard, Manhattan
`John Shine, Canberra
`Joan A. Steilz, New Haven
`Robert T. Tjian, Berkeley
`Michael Wigler. Cold Spring Harbor
`Patricia Zambryski. San Francisco
`
`Editorial Associates
`
`Barbara Cordell. Boston
`David D. Moore, Boston
`
`Pu blisher
`
`Alan M. Edelson, Ph.D., Raven Press.
`New York
`
`Publication and Advertising Staff
`David A. Christie, Production Manager.
`Raven Press. New York
`Nancy Gordon, Assistant
`Editor-in-Chief, Boston
`Irene Rubinson, Advertising Manager.
`Raven Press, New York
`
`to the
`
`Volume 1, Number 4, 1982
`
`ilomudlul Librlly ucsp
`
`CONTENTS
`
`Genetic Evidence for Separate Functional Domains
`on the Human Adenovirus Specified, 72kd, DNA
`Binding Protein
`Daniel F. Klessig and Margaret P. Quinlan
`
`Regulation In Vivo of a Cloned Mammalian Gene:
`Cadmium Induces the Transcription of a Mouse
`Metallothionein Gene in SV40 Vectors
`
`Dean H. Hamer and MaryJane Walling
`
`Use of a lac Promoter-Operator Fragment as a
`Transcriptional Control Switch for Expression of
`the Constitutive lpp Gene in Escherichia coli
`Kenzo Nakamura, Yoshihiro Masui, and
`Masayori Inouye
`
`An Analysis of mRNAs for a Group of Heat Shock
`Proteins of Soybean Using Cloned cDNAs
`Fritz Schoffl and Joe L. Key
`
`Molecular Cloning of Rhizobium trifolii Genes
`Involved in Symbiotic Nitrogen Fixation
`K. F. Scott, J. E. Hughes, P. M. Gresshoff,
`J. E. Beringer, B. G. Rolfe, and J. Shine
`
`Transformation of Mammalian Cells to Antibiotic
`Resistance with a Bacterial Gene Under Control
`
`of the SV40 Early Region Promoter
`P. J. Southern and P. Berg
`
`(Continued on next page)
`
`This journal is listed in Current Contents.
`
`Journal of Molecular and Applied Genetics (ISSN 0271-6801) published bimonthly by Raven Press, Publishers, 1140 Avenue of the
`Americas, New York. N.Y. 10036. Subscription ratesfar Vol. I. l98l/2: Personal subscriptions $80 in U.S., $90 elsewhere; institutional
`subscriptions $135 in U.S.. $145 elsewhere. Air delivery included for European and Mediterranean countries: for air delivery elsewhere.
`add $15. Payment should accompany all orders. Please enclose present mailing label with all change of address requests. Address
`advertising inquiries to Advertising Manager, Raven Press. Copyright © 1982 by Raven Press.
`When citing this journal, abbreviate as J Mol Appl Genet.
`
`Genzyme Ex. 1004, pg 58
`
`

`
`_;.mrrml rrf.\!uI.-rulur uml Appllwl (ivmllr .\
`1;_tZ7—J-3|
`'t'v I981 Raven Press. New Yurk
`
`Transformation of Mammalian Cells to
`
`Antibiotic Resistance with a Bacterial Gene Under
`Control of the SV40 Early Region Promoter
`
`P. J. Southern and P. Berg
`
`D('[)IlI‘II)It’I1I u_/'Bim'livn1i'.r!rv, Stanford UniI'w'.viI_\' M¢'di<'u/ Ce/Her, S!uufm't/. Ca/t_'forni(I. U..S'./1.
`
`Summary: A bacterial gene (neo) conferring resistance to neomycin-
`kanamycin antibiotics has been inserted into SV40 hybrid plasmid
`vectors and introduced into cultured mammalian cells by DNA trans-
`fection. Whereas normal cells are killed by the antibiotic G418, those
`that acquire and express neo continue to grow in the presence of G418.
`1n the course ofthe selection, neo DNA becomes associated with high
`molecular weight cellular DNA and is retained even when cells are
`grown in the absence of G418 for extended periods. Since neo provides
`a marker for dominant selections. cell transformation to G418 resis-
`tance is an efficient means for cotransformation of nonselected genes.
`Key Words: Antibiotic resistance—Cell
`transformation—DNA
`transfection—Recombinant DNA—Bacterial genes.
`
`There are two principal approaches available
`for the introduction of exogenous DNA into
`mammalian cells. Simian virus 40 (SV40) can
`be used as a transducing vector because it can
`replicate vegetatively in primate cells (1) or be-
`come integrated into host chromosomal DNA
`in a wide variety of cells (2). The experimental
`strategy has been to replace various regions of
`the viral genome with cloned segments of DNA
`and to propagate the recombinants with the aid
`of helper viruses in cultured animal cells
`(3-10). Alternatively. exogenous DNA may be
`introduced directly into recipient cells by either
`the calcium phosphate precipitation technique
`
`Reccivcdlanuary 4. l982:acccpted February 18. 1982.
`Address correspondence and reprint requests to Dr.
`P.J. Southern. Department of lmmunopathology.
`’5:ripps Clinic and Research Foundation, La Julla. CA
`.037.
`
`(11), DEAE-dextran (12). or microinjection
`(13.14). The pioneering experiments for this
`approach relied on the transformation of TK'
`mammalian cell
`lines to a TI(’' phenotype
`with the herpes simplex thymidine kinase gene
`(15-17). Subsequently. unrelated DNA se-
`quences have been integrated with the thy-
`midine kinase gene in either linked or co-
`transformation experiments (18,19). Transfor-
`mation of appropriate mutant cell lines has also
`been demonstrated with cellular DNA se-
`
`quences and this approach has allowed the
`isolation of the chicken thymidine kinase gene
`(20) and the hamster adenine phosphoribosyl
`transferase gene (21). Morphologic transfor-
`mation of normal cells forms the basis of cur-
`
`rent attempts to isolate cellular oncogenes
`(22.23).
`Unfortunately. experiments that rely upon
`complementation of cell mutations by trans-
`
`Genzyme Ex. 1004, pg 59
`
`

`
`338
`
`P. .I.
`
`.S'()l/Till-.'RN AND P. b‘I:’RG
`
`-
`
`duced genes are limited by the availability of
`mutant mammalian cell types to serve as gene
`recipients. Dominant-acting genetic markers.
`for example.
`those that produce a selectable
`change in the phenotype of normal cells. offer a
`solution to this difficulty. The isolation of
`methotrexate—resislant
`transformants after
`transfection of normal cells with DNA from
`drug-resistant cells (24) exemplifies this ap—
`proach. However.
`transformation for metho-
`trexate-resistant dihydrofolate reductase is in-
`frequent and. therefore. the utility for cotrans-
`formation with other genes appears limited.
`Recently. our laboratory devised a family of
`SV40 hybrid plasmid vectors to facilitate
`studies of gene transfer and gene expression in
`mammalian cells (8.25). Plasmids containing
`DNA segments coding for rabbit ,8-globin (25),
`mouse dihydrofolate reductase (10). and E. call‘
`xanthine-guanine phosphoribosyl transferase
`(8) can induce the synthesis of the corre-
`sponding gene products in mammalian cells.
`The expression ofthe bacterial gene, gpt. per-
`mits the utilization of xanthine as a substrate
`for the purine salvage pathway and a selection
`can be established in which gpt functions as a
`dominant marker for cell transformation (26).
`ln this paper, we describe a second bacterial
`gene which. when incorporated into the same
`family of plasmid vectors, also provides a
`dominant selective marker for transformation
`of cultured mammalian cells.
`The selection for transformation of mamma-
`
`lian cells relies on cell killing by an amino-
`glycoside antibiotic. G418 (27). The structure of
`G4l8 resembles gentamicin, neomycin. and
`kanamycin (28) but. unlike these related com-
`pounds, G4l8 interferes with the function of
`80S ribosomes and blocks protein synthesis in
`eukaryotic cells (27). These aminoglycoside
`antibiotics can be inactivated by the bacterial
`phosphotransferases, Al’H(3')l| and APH(3’)l
`encoded by transposons Tn5 and Tn60l,
`re-
`spectively (29). Jiminez and Davies (30)
`showed that yeast could be genetically trans—
`formed to G418 resistance by the phospho-
`transferasc gene contained in Tn6tll DNA.
`Thus. it seemed feasible (suggested by Sydney
`
`J. /Wnl. /1/:/J/. (p'r'Iu'l.. Vol. I. No. 4. [U82
`
`Brenner) that the acquisition and expression 0
`the phosphotranst'erase gene by mammaliar
`cells might confer rrcsistance to G4l8 toxicity
`Accordingly.
`the phosphotransferase gent
`from Tn5 (designated here neo) (29.3l) was in-
`troduced into the mammalian transcription unit
`of the pSV plasmid vectors.
`In this arrange-
`meat the SV40 early promoter is 5'-proximal.
`and an intron and polyadenylation signal are
`3'-proximal.
`to the neo gene (8.25). Transfec
`[ion of a wide variety of mammalian cetl lines
`with these pSV-neo recombinants yields stable
`transformants that are resistant to G-H8 at a
`frequency of one transformant per I0‘-I0’
`transfected cells. Our data suggest that trans
`formation results from the acquisition. mainte«
`nance. and continued expression of the neo
`gene in the cellular genome. Concurrent with
`our experiments. Colbére-Garapin ct al.
`(32)
`achieved mammalian cell
`transformation to
`G418 resistance with recombinant DNA con-
`taining the Tn5 neo gene linked to the herpes
`thymidine kinase promoter DNA segment.
`
`I MATERIALS AND METHODS
`
`Cells
`
`A wide range of cultured mammalian cells
`are sensitive to the antibiotic G413 (Table ll
`and several of these have been used for trans-
`
`formation experiments. The cells were rou-
`tinely maintained in Dulbecco—modified Eagle
`medium containing l0'7c newborn calf serum.
`penicillin and streptomycin. and the indicated
`concentrations of G418 were added to lhc
`medium.
`
`Enzymes
`
`Restriction enzymes were purchased from
`New England Biolabs and Bethesda Research
`Laboratories and digestions were performv-‘J
`according to the supplier's specifications. T4
`polynucleotidc kinase was purchased from
`New England Nuclear and SI nuclease frttll‘
`Boehringer Mannheim. T4 DNA ligase and fi-
`mli DNA polymerase I were kindly provided
`by S. Schcrcr. Stanford University.
`
`Genzyme Ex. 1004, pg 60
`
`

`
`'l'RAN.S'l-'(JRi\IA 7l()lV ‘/0 »tN7'IBl()'l'l(‘ R[;'SI.S"l‘/tNC'E
`
`329
`
`Antibiotic G418
`
`Samples of antibiotic G418 were generously
`provided by Dr. P. J. L. Daniels of Schering
`Corporation. Stock solutions containing 4
`mgjml G418 in 1()() mM N-2-hydroxyethylpi-
`pcrazine-N’-2’—ethanesulphonic acid buffer, pH
`7_3_ were stored at —20°C and added in ap-
`proprltlle amounts to the cell culture medium.
`The G418 concentration refers to the actual
`amount of drug in the solution and takes into
`account that the solid material was only 40-
`509? G418.
`
`DNA Transfection and Selection of
`Transformed Cells
`
`Supercoiled plasmid DNA, without added
`:arrier DNA. was introduced into tissue cul-
`ure cells (10 tag for approximately 5 x 10"
`:ell5) using the calcium phosphate precipitation
`echnique (11) with the addition of a glycerol
`il10Cl( after 4 h (33). About 48 h after exposure
`0 DNA.
`the cells were trypsinized and re-
`ilated at a 1:20 dilution. Within 12- 16 h, G418
`was added to the medium at a concentration of
`
`100 ayml. The medium plus drug was changed
`:very 4 to 5 days. Colonies were first detected
`ifter about 7 days in the selective medium and,
`1-13 days later.
`independent colonies were
`rypsinized in cloning cylinders and transferred
`0 microtiter wells. When the colonies were
`
`.mall. the transplanted cells were grown non-
`clectively for an initial 2-3 days. Once estab-
`ished, the clones were expanded to stable cell
`ines in medium containing 400 pg/ml G418. In
`.onie instances.
`the initial selection and sub-
`:loning used 400 pg/ml of G418 but the trans-
`ormed cells were subsequently maintained in
`‘.00 pg/ml of G418.
`The selection strategy of permitting cell
`zrowth prior to the addition of G418 was
`idopted because a significant reduction in the
`ransformation frequency occurred if G418 was
`I(.l(.lCt.l before 48 h. The tragsfected cells were
`eplated at lower cell density because G418 is
`nost effective against dividing cells. Con-
`cquently.
`if cells become stably transformed
`
`early after transfcclion. cell division prior to
`selection may result in overestimation of the
`transformation frequency. Nevertheless. none
`of the transformants from randomly selected
`colonies appeared to have the same organiza-
`tion of the integrated pSV-neo DNA.
`
`Analysis of Transformed Cell DNAs
`for pSV-neo Sequences
`
`High molecular weight cellular DNA was
`extracted as described by Wigler et al. (17),
`incubated with an excess of restriction en-
`
`zyme, and the digests were separated by elec-
`trophorcsis in 0.8% agarose gels. After a mild
`depurination reaction (34).
`the DNA was
`transferred from the gel
`to diazobenzyloxy-
`methyl paper (DBM paper) (35), hybridized
`with radioactively labeled DNA probes (36),
`and radioautographed using Kodak XR5 film
`and Cronex lightning fast intensification screens
`at -70°C (37).
`
`Analysis of Cytoplasmic RNA
`Extracted from pSV2-neo
`Transformed Cells
`
`Cytoplasmic RNA was extracted from
`'semiconfluent cultures of transformed cells as
`
`described previously (38). The RNA was sepa-
`rated from contaminating DNA by pelleting
`through cesium chloride and then the poly A*
`RNA fractions were characterized using the
`Weaver-Weissmann variation (39) of the
`Berk-Sharp procedure (40). DNA hybrization
`probes (shown with individual experiments)
`were prepared by labeling appropriate restric-
`tion fragments at
`their 5'-ends with [y-MP]-
`adenosine triphosphate and polynucleotide
`kinase (41). The DNA probes were hybridized
`with RNA samples under conditions of DNA
`excess. RN A-DNA hybrids were digested with
`S1 nuclease, and the protected fragments were
`analyzed by gel electrophoresis (39,40).
`
`Protein Labeling and
`lmmunoprecipitation Reactions
`
`Semiconfluent plates of pSV2-neo trans-
`formed cell lines were labeled for 14 h at 37°C
`
`J. Mu/. /lppl. (ft'Iu‘I., Vul. I. No. 4. W33
`
`Genzyme Ex. 1004, pg 61
`
`

`
`_t’_t’()
`
`P. J. .S'()UTIII:'RN AND I’. BERG
`
`with ["'H]leucine (200 p.Ci/plate. specific activ-
`ity 55 CL’mmol. New England Nuclear Labo-
`ratory). The soluble proteins were extracted
`from approximately 2 X 10’ cells (7) and im-
`munoprecipitated with an antiphosphotransfer-
`ase APH(3’)ll serum (provided by J. Davies.
`Geneva). Est-/iw'icIiiu wli cells (HB10l) con-
`taining plasmids were grown to approximately
`2 X I0“ cells/ml in M9 minimal medium plus
`glucose with supplements of leucine. proline,
`threonine. and thiamine. Samples ofthe cultures
`(0.5 ml) were washed and resuspended in the
`same medium lacking leucine and then [“H]leu-
`cine was added (200 )uCi/ml) for 60 min at 37°C.
`Excess unlabeled leucine was added and. after
`washing in M9 medium, the cells were disrupted
`by sonication. Cell debris was removed by cen-
`trifugation at l4,000g for 10 min and the super-
`natant was used directly for immunoprecipitation
`reactions. After incubation overnight at 0°C. the
`immune complexes were adsorbed to inacti-
`vated S. aurens cells (IgGsorb, Enzyme Cen-
`ter. Boston) and removed by centrifugation
`(42). The S. uureus cells were washed exten-
`sively and the bound proteins were eluted and
`electrophoresed in SDS polyacrylamide gels
`(43). After electrophoresis,
`the gels were
`treated with EN“!-IANCE (New England Nu-
`clear Laboratory). dried and autoradiographed
`as described above.
`
`RESULTS
`
`Mammalian Cells Are Sensitive to G418
`
`The sensitivity of various cultured cell lines
`to G418 was assessed by plating cells at
`low
`cell density in microtiter wells in a medium
`supplemented with various concentrations of
`G418. Even at the highest drug concentration
`tested (800 pgjml), sensitive cells divided once
`or twice before cytotoxicity was observed. The
`response time for cell killing appears to corre-
`late with growth rate. since the most rapidly
`growing cells are killed in the shortest inter-
`vals. _At
`lower concentrations of G4|8 (lt)()
`p.g/ml) there is a significant delay but the cells
`are killed eventually. All of the cell lines that
`
`J. Muf. App], (it'm'!.. Vol. I. NH. 4. I982
`
`TABLE I.
`
`(I-II8-.t't'n.t’iriiw nitmmmlhui ('1',/it-Il(.\"'
`
`Monkey
`
`(‘V1
`CVI-P
`TC7
`COS
`
`Human
`
`l.NSV
`HeLa
`l-(-562
`
`Mouse
`
`L
`Llk
`3T3
`3T6
`PCC4
`F9
`MEL
`
`"So far. no cell
`resistant to G-H8.
`
`line has been |'ound that is naturally
`
`have been tested (Table l) are killed by G-(I8
`but CV] and HeLa are unusual because. at
`
`high cell density, these cells may require 10- I4
`days in G418 (400 iiymll before the cell killing
`can be observed.
`
`Construction of Recombinant
`
`Plasmids Containing neo
`
`The bacterial transposon Tn5 encodes a gene
`(neo) whose protein product—a phosphotrans-
`ferase (APH(3')Il)—confers resistance to the
`
`kanamycin-neomycin group of antibiotics (31).
`From the studies of the organization of Tn5
`DNA. Reznikoff and colleagues (44.45) were
`able to identify the DNA segment that is essen-
`tial for the expression of neo. The CoiEl:Tn5
`plasmid pRZi l2 (44). a deleted form of the or-
`ginal Col El:Tn5 hybrid plasmid (pRZl02. Fig-
`l). was the source of the neo DNA segment
`pRZl I2 DNA was digested to completion with
`Hincll endonuclease and the 3.5 kl) neo DNA
`
`segment was obtained by agarose gel electro-
`phoresis. After ligating a decanucleotide se-
`quence containing the BumHl restriction silfl
`(Collaborative Research) to the ends ofthe n60
`
`segment (46) the mixture was digested with an
`excess of Bu/nHl and I-Iindlll restriction 8"‘
`donucleases and the resulting |.4 kb neo DNA
`fragment was purilied by gel clcClI'0ph0resi.\-
`This fragment. containing Ilindlll and BumHl
`cohesive ends at the 5’- and 3'-ends, respc“
`tively. was inserted between the Hindlll and
`BumHl restriction sites in pBR322 DNA. Thc
`resulting plasmid. pBR—neo (Fig. I). confers rc-
`sistancc in IL’. cult‘
`to both ampieillin and
`
`Genzyme Ex. 1004, pg 62
`
`

`
`’IRANSl-'0R.tM 'I‘l()N '10 AN'I‘lBI()7‘l(‘ Rl;‘SIS'I'AN(‘I;'
`
`331
`
`donuclcasc digestion and substituting thc neo
`fragment via the corresponding cohesive ends.
`The pSV3-nco and pSV5-nco (Fig. 2) deriva-
`lives were constructed from pSV2-neo as pre-
`viously described (8.25). Each of the pSV-neo
`plasmids replicates efficiently in E. to/i strain
`HBIUI and confers resistance to ampicillin and
`neomycin. Cloned isolates of each of the re-
`combinant plasmids were shown to have the
`anticipated structures by appropriate restric-
`tion enzyme analyscs (data not shown).
`
`Cell Transformation with Recombinant
`
`Plasmids Containing neo
`
`Transfections of Ltk' cells with pRZll2 or
`pBR-neo plasmid DNA yielded occasional
`G418-resistant colonies (a frequency of about
`one transformant in 5 X 10" transfected cells).
`However, with 3T6 cells as recipient no trans-
`formants have been recovered with pRZll2
`DNA and only one transformant has been iso-
`Iated after transfection with pBR-neo (fre-
`quency about one transformant
`in 10‘
`trans-
`fected cells).
`In control experiments, cells
`transfected with either pSV2-gpt (8), pSV2-BG
`(25) DNA, or mock-transfected without DNA
`have never yielded G418-resistant colonies
`(frequency less than I
`transformant in [07
`transfected cells).
`In contrast to the low frequencies of G418-
`resistant transformation with pRZl12 (ColEl-
`neo) or pBR-neo plasmid DNAs, several dif-
`ferent mammalian cell lines were transformed
`
`to G418 resistance at relatively high frequency
`with the pSV-neo plasmid derivatives (about
`one transformant in 10‘
`to 10"’ transfected
`
`cells). Although the transformation frequencies
`with the different pSV-neo recombinants fall
`within a relatively narrow range." there is a con-
`sistent small difference which reflects the
`
`plasmid's potential for replication in different
`host cells (Table 2]. For example.
`the fre-
`qucncy of stable G418-resistant monkey cell
`transformants is two- to three-fold lower with
`
`pSV3-neo than with pSVS—nco. whereas the
`converse applies to the formation of G418-
`resistant mouse cell transformants. This differ-
`
`J. Mal. App]. (:'wtr'I.. Vol. I. No. -I.
`
`IUR3
`
`H/mil II
`
`l
`Bglll
`pnztoz
`
`Hn.:I
`
`Hi/ttllll
`
`SJ”
`55””
`EA1"G
`l<— neo -9!
`
`-I
`
`HI)-ll
`
`IHHICHI Hm.’/III
`H) J.) I
`Bull I
`
`- ‘
`
`Hmrl I
`I
`I
`iAT6
`i<— neo —>t'
`
`pRz1 12
`
`1 Hiucll CLEAVAGE
`
`Hmrll II
`
`l_—jl..jl
`Hmcll
`|Eglll
`Hi’/tcll
`
`BamHl LINKERS
`B.mtHl + Hintllll CLEAVAGE
`ISOLATION OF neo GENE FHAGMENT
`
`Him‘/I ll
`‘Salli
`
`tl:l.:mHI
`
`INSERTION INTO pen322
`SELECTION FOR Amp"‘,~eoR
`
`W
`
`Hinr/I II
`
`ilc. 1. Organization of transposon Tn5 and scheme for the
`onstruetion of recombinant plasmid p8Ft-neo. The plasmid
`tFtZt02 contains a complete copy of Tn5 inserted into ColE1.
`'he inverted repeat sequences at the ends of the transposon
`;re shown as thick lines. The region essential for expression
`tl neornycin resistance in E. coli (neo) is indicated together
`lllh the ATG codon that initiates the coding sequence of the
`thosphotranslerase. Flelevant restriction endonuclease rec-
`tgnttion sites are included in the diagram. pRZ112 was de-
`tved from pFtZ102 by partial digestion with Hincll endonu-
`tease and ligation to eliminate a large segment of Th5 DNA to
`ne 3‘ side at neo (Hinc|I endonucleasa cleaves at Salt and
`‘pat endonuclease recognition sites.) Details of the ma-
`ipulations involved with the construction of pBR-neo are
`liven in the text.
`
`tcomycin: since the neo DNA segment inter-
`upts the tetracycline resistance gene, cells
`arrying this plasmid are sensitive to this anti-
`iiotic.
`-
`
`The cloned neo DNA segment was readily
`ntroduced into the plasmid vector—pSV2 (8)
`Fig. 2) by excising the B-globin cDNA segment
`rom pSV2-}3G (25) with Hindlll and Bglll en-
`
`Genzyme Ex. 1004, pg 63
`
`

`
`I’. J. .S'()UTl/ERN AN!) I’. B!-IRG
`
`pBR.’l22 on
`
`panazz on
`
`989322 an
`
`pSV3- Mo Eamhfl
`
`FIG. 2. Structures of the psv-neo hybrid ptasmids. The psv plasmids are composed of DNA segments from various source
`oBFi322 DNA. represented by the solid black are. contains the pBFl322 origin 01 DNA replication (pBFi322 on) and the ,6-Iactama
`gene [Arrtp"}: the hatched segment represents the new gene [1 4 kb fragment. Fig. t);SV-10 DNA sequences are indicated as II
`dolled Seqmflnlsv The 5W0 Ofléilfi 0! DNA replication (SWO on) and the SV40 early promoter are present on a small fragme
`[SWO map UNIS 0.'a"l ~D.B5] immediately 5' to the neo segment. The plasmids pSV3-neo and pSV5-neo were constructed lro
`pSV2-neo by insertion ol either an intact SWO early region or an intact polyoma early region that contains a duplication oi ti
`viral ongin oi‘ replication [8].
`
`once may be related to the ability of pSV3
`plasmids to replicate in monkey cells and pSV5
`plasmids to replicate in mouse cells (25). A
`comparable result was previously noted in
`transformations with pSV3-gipt and pSV5-gpt
`recombinants (26).
`
`Stable Integration of neo DNA Sequence:
`after Transfection into Cultured Cells
`
`The content and organization of neo DH.‘
`sequences have been examined in a number 0
`stable G4l8 transformed cell lines. The Llk’ 0
`
`TABLE 2. Trun_\_/in'muIi'mi _/i't'([m‘7l('l'('.\' flu‘ I'¢'(‘0mbiItmtl'
`Hon phi.\'Iiti(I.\"‘
`
`Host cells
`
`Ltk
`
`3T0
`
`'l‘C7
`
`I X [H '
`7 X ll)
`~3 X I0 ‘
`pSV2-ncu
`6 X It)
`-"
`'
`|.S x Ill
`~-3 X ll)
`'
`pSV3-neo
`2 x |0 '
`'
`4 x It)
`-3 X It)
`'
`pSV5-nco
`No colonies isultilcd
`I X ll) ‘
`5 X It) “
`pBR-neo
` {_
`
`" ’l‘rzinsl'm'muIiun frequency is cxprcsscil us the lrtiction ul'ci.-lls plated
`that product: viaihlt: colonies in selective medium Following ll’illl.\'rC(.'li0l‘l
`with siituruling Icvcls oi" IJNA.
`
`J. Mill.
`
`.-lmil. (:'i'm'I.. HII. I, No. J, [982
`
`Genzyme Ex. 1004, pg 64
`
`

`
`TR/l NS FORMA TION TO A NTIBIO TIC‘ RESIS'l'A N('l;'
`
`1Tb transformants arising from translections
`;\'i[h pSV2-neo. pSV3—nco. or pSV5—neo do not
`contain p|asmid—rclated sequences (limits of
`detection 0.5 plasmid molecules per cell) in the
`low molecular weight DNA of a Hirt superna-
`tant (47l. However. cell DNA obtained from
`0418-resistant transformants contained nucle-
`uridc sequences homologous to the transfecting
`plasmid DNA. High molecular weight DNA
`from a representative set of pSV2-neo Ltk‘
`transformants was cleaved with EcoRl restric-
`tion endonuclease. electrophoresed in an
`agarose gel.
`transferred to DBM paper. and
`then hybridized with radioactively labeled
`pSV2-neo DNA (Fig. 3). Since pSV2-neo DNA
`contains a single Er'0Rl restriction site, each
`integrated plasmid copy should produce two
`hands after hybridization with the pSV2-neo
`probe if there is no rearrangement or serum-
`bling of the plasmid DNA sequences during in-
`tcgralion. Most of the transformants that have
`been examined appear to have a low plasmid
`copy number, generally one to five copies per
`cell. Transformant 2G is exceptional
`in that
`there are multiple copies of the plasmid and a
`substantial amount of apparent
`linear pSV2-
`neo DNA is produced by digestion with E¢'oRl
`endonuclease (Fig. 3). Since no circular plas-
`mid DNA was present in the Hirt supernatant
`fraction prepared from 2G cells and the high
`molecular weight DNA of the Hirt pellet hy-
`bridized strongly to a radioactively labeled
`pSV2-neo probe (results not shown). it is likely
`that transformant 2G contains either a tandem
`
`array of integrated plasmid DNA or autono-
`mously replicating polymeric plasmid DNA.
`Although the latter possibility can not be
`excluded.
`it seems less likely because the
`G418-resistant phenotype as well as the copy
`number and organization of the neo DNA se-
`quences do not change after passaging in non-
`selective medium (see below).
`The Ltk" 2F and 20 cell
`
`lines have been
`
`cultured for over 3 months (approximately 100
`cell generations) in medium lacking or con-
`laining G418 (200 ,ug/ml); such cells remain
`fully resistant to G418 when challenged in
`
`Ltk‘/pSV2neo
`/
`
`IZFIZGIZJI
`
`‘MI
`
`‘
`
`UNEAR
`_ pSV2neo
`
`FIG. 3. Detection of pSV2-neo DNA sequences in trans-
`formed cells. High molecular weight cell DNA was extracted
`lrom representative G418-resistant Ltk' cell lines that had
`been transformed with pSV2-neo plasmid DNA. The DNA
`samples were digested with an excess of Eco RI restriction
`endonuclease and then fractionated by electrophoresis in a
`0.8% aglarosa gel. DNA In the gel was transferred to DBM
`paper and hybridized with radioactively labeled pSV2-neo
`DNA. There is a single recognition site in pSV2-neo DNA for
`Econ! cleavage and the marker track (M) shows the position
`oi pSV2-neo linear DNA. Approximate molecular sizes were
`derived trom A DNA restriction fragments in an adjacent slot
`on the gel.
`
`selective medium. The relative plating eff-
`ciency ofthe 2F or 2G cell lines after seeding in
`the presence or absence of G418 is within ex-
`perimental variation,
`the same (ratio 0.6 to
`0.9:l.0). Moreover.
`there was no discernible
`variation in the organization of the pSV2-neo
`DNA in the high molecular weight cell DNA
`obtained from 2F and 2G cell DNAS recovered
`
`times during their growth in the
`at different
`presence or absence of G418 (Fig. 4). Thus, it
`appears that
`the amount and arrangement of
`the pSV2-neo plasmid DNA sequences are sta-
`
`J. Mal. r|[‘[li. Cr‘Ilr'l., Vol. I. No. 4. I93.’
`
`Genzyme Ex. 1004, pg 65
`
`

`
`P. J. SOUTHERN AND P. BERG
`
`Ltk‘ pSV2neo2F
`as-T
`‘b
`-3*
`ti‘
`ta
`,0
`,°
`
`1
`
`'3
`
`I
`
`L tl-t " pSV_2neo2G
`’jja._j_.,
`'5
`\
`/G‘|
`
`/
`5'3’
`V8. I X0 I
`
`o
`W l r
`
`Ltk‘pSV2neo2G
` ‘
`'3’
`'3’
`_e"
`,<>°'
`
`'1?’
`
`FIG. 4. Detection of pSV2-neo DNA se-
`quences alter extended passaging ol
`transtormed cells. High molecular weight
`chromosomal DNA was extracted lrom
`parallel cultures of the lranslormed cell
`lines 2F and 26 that had been maintained
`in the presence (+G-116) or absence
`(—G418) ol G418 (see text). DNA samples
`were digested with EcoRI endonuclease
`and analyzed as described in Fig. 3. The
`tracks at the extremities ol the gel
`(labeled 2F and 26) contain samP|8S 0i
`the original DNA preparations (see Fig. 3).
`The central track contains parental Ltk’
`cell DNA. The panel at the right ot the tig-
`ure is a shorter exposure during au-
`toradiography ot the Ltk‘ pSV2-neo 2G
`cell DNA.
`
`ble during growth of these two transformed cell
`lines under selective or nonselective condi-
`tions.
`
`We sought to determine if stable transforma-
`tion for the transduced marker could occur in
`the absence of selection. Accordingly, semi-
`confluent cultures of mouse 3T6 cells were
`transfected with pSV3-neo or pSV5-neo DNA
`and one set of cells was maintained under the
`standard conditions for transformation and an-
`other set was propagated in the absence of
`G418 for about
`l2 generations (2 weeks) and
`then the G418 selection was applied. The
`transformation frequency in the first set was
`I0“ to 10"" whereas the second set yielded
`about one—fifth the number of transformants
`predicted from the initial value and the number
`of cell divisions that had intervened before the
`selection was applied. Assuming that
`trans-
`formed and normal cells divide at the same rate
`in medium lacking G418 and considering the
`inherent
`inaccuracies of the experiment.
`it
`
`I. Moi. A/I/II. (ir'm'I.. Vol. I. No. 4. I982
`
`seems that the neo marker is associated will
`the cellular genome relatively early and is re
`tained in the absence of G418 selection.
`
`Expression of the neo Gene in pSV2-neo
`Transformed Cells
`
`The expression of neo in G418-resistant
`transformants has been confirmed by the de-
`tection of neo mRNAs and phosphotransfcrase
`protein. Cytoplasmic. polylA)* RNA was iso-
`lated from pSV2-neo transformed Ltk‘ cells
`(isolates F and G) and hybridized to an end-
`labeled DNA probe specific for pSV2-neo se-
`quences.
`In each sample. digestion with SI
`nuclease and electrophoresis of the protected
`DNA in an agarose gel (39.40) yielded a single
`fragment of I500 bases (Fig.5). The production
`of 21 I500 base fragment is consistent with thc
`occurrence of an RNA that extends throughout
`the length ofthe neo segment and suggests that
`
`Genzyme Ex. 1004, pg 66
`
`

`
`IIi’xt.'\’.5'I-'()R.'tI.it"HUN H)AN’!/II/()7l('RESIS1}tNCl£
`
`MARKERSax
`
`FIG. 5. Detection of neo sequences in cyto-
`plasmic FINA extracted from pSV2-neo trans-
`formed Ltk' cells. A DNA probe was prepared by
`5' end-labeling with "P at the Baml-tl restriction
`site at the 3’ extremity oi the neo DNA fragment.
`(The BamHI site was not preserved in the con-
`struction ot pSV2-neo and the probe for this ex-
`periment was prepared trom another derivative
`plasmid.) Relevant restriction endonuclease
`cleavage sites are indicated in the diagram.
`RNA-DNA hybrids were formed under condi-
`tions of DNA excess (40) and the S1 nuclease
`digests were lractionated on a 1% neutral
`agarose gel. Marker bands were produced by
`digesting the DNA probe with Bgll, Hindlll and
`Bglll restriction endonucleases. The DNA frag-
`ment protected by the transformed cell FtNAs is
`shown in the diagram as a continuous line
`under the representation ot the DNA probe.
`
`PROBE FOR pSV2-neo RNA
`
`EcoFt|
`
`Pirull
`
`Hgfl Him/Ill
`
`Ball!
`
`|—/
`
`290
`
`72
`
`320
`
`Bamfll
`
`OBSERVED PROTECTED
`FHAGMENT
`
`1500
`
`the 5'-end of the RNA is located beyond the
`Hiiidlll site. most probably in proximity to the
`SV40 early promoter. Note that although the
`copy number of pSV2-neo DNA sequences is
`much higher in the 2G cells (Figs. 3 and 4), the
`amount of neo mRNA in the 2F and 2G trans-
`tnniicd cell lines is about the same.
`"Io determine if the Ci-H8-resistant transfor-
`
`m..nts produce phosphotransferase protein.
`scniiconfluent cultures of normal and trans-
`lormed cells were labeled for l4 h at 37°C with
`lllleucine. Labeled extracts of E. Coli har-
`Nring pBR-neo or pBR322 were made for
`mmpurison (see Materials and Methods). Im-
`munc complexes prepared from each of these
`c\ir.ii.‘ts were clectrophoresed in sodium dode-
`cyt sulphate polyacrylamidc gels (Fig. 6). It is
`fvidcnt that the immunoprecipitate from mli
`"fitting pBR-neo contains a peptide of 25
`Ktiodilllttns. whereas the extract from cells
`"3-int! neo does not contain that peptide. The
`
`molecular weight estimate for APH(3’)lI ob-
`tained from E. coli agrees with earlier findings
`(48). Comparisons of the electrophoretic pat-
`tern of immune complexes obtained from un-
`transformed and two pSV2-neo transformed
`Ltk‘ cells (isolates F and G) reveal that the
`transformants contain a specific band corre-
`sponding to a protein of 28 kilodaltons with no
`indications of a protein of 25 kilodaltons. This
`difference in apparent molecular weight be-
`tween the immunoprecipitated APH(3’)Il
`found in E. cult‘ and the transformed mouse
`cells is considered in the Discussion section.
`
`Although the copy number of neo DNA is con-
`siderably greater in isolate 2G than 2F,
`the
`amount of phosphotransferuse peptide appears
`to be about the same. even somewhat greater in
`‘JP:
`the same disparity occurs between neo
`DNA copy number and neo mRNA (see Fig.
`5). This suggests that many of the neo genes in
`isolate 2G may not be expressed.
`
`J. Moi. .-1]!/VI. Gt‘Hi'I.. \'u/. I. Nil. 4. WI"!