~
`
`v
`
`I
`
`;~ !
`
`I
`
`I
`1..
`, , c :?.,
`"' I
`~\
`I,
`
`~ I
`
`I
`
`I
`
`I
`
`10LlJMF l· NlJMBER 4·l9R·?·RAVEN PRESS
`
`Sanofi/Regeneron Ex. 1 004, pg 62
`
`Merck Ex. 1004, pg 62
`
`

`
`Journal of
`Molecular and Applied Genetics
`
`Editor-in-Chief:
`
`Howard M Goodman, Ph.D.
`Department of Molecular Biology
`Massachusetts General Hospital
`Boston, Massachusetts 02114, U.S.A.
`
`RECEIVED
`JUL 14 1982
`
`Volume 1, Number 4, 1982
`
`llomedlca~l l.ibr•ry UCSD
`
`1
`
`CONTENTS
`
`263 Genetic Evidence for Separate Functional Domains
`on the Human Adenovirus Specified, 72kd, DNA
`Binding Protein
`Daniel F. Klessig and Margaret P. Quinlan
`
`273 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
`
`289 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
`
`30 I An Analysis of mRNAs for a Group of Heat Shock
`Proteins of Soybean Using Cloned cDNAs
`Fritz Schorn and Joe L. Key
`
`315 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
`
`327 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 Curren/ Contents.
`
`Editorial Board
`
`John Abelson, La Jolla
`frederick Ausubel, Cambridge
`John R. Bedbrook, Canberra
`Lawrence Dogorad, Cumbridge
`Mary-Dell Chilton, Sr. Louis
`Richard A. Flavell, London
`Raymond F. Gesteland, Salt Lake City
`Gary Gussin, Iowa City
`Dean H. Hamer, Bethesda
`Masayori Inouye, Stony Brook
`Robert Kamen, London
`\'uet Wai Kan, San Frc111cisco
`Laurence H. Kedes, Palo Alto
`Daniel Klessig, Salr Lc1ke City
`Thomas Maniatis, Cambridge
`David W. Martin, Jr., San Francisco
`Marc Van Montagu, Gem
`Richard C. Mulligan, Cambridge
`Alexander Rich, Cambridge
`JctT Schell, C ologm•
`James Shepard, Manhattan
`John Shine, Canberra
`Joan A. Steitz, New Ha~en
`Robert T. Tjian, Berkeley
`Michael Wigler, Cold Spring Harbor
`Patricia Zambryski, San Francisco
`
`Editorial Associates
`Barbara Cordell, Boston
`David D. Moore, Boston
`
`Publisher
`Alan M. Edelson, Ph.D., Raven Press,
`New York
`Publication and Advertising Staff
`David A. Christie, Production Manager,
`Raven Press, New York
`Nancy Gordon, Assisianl to the
`Editor-in-Chief, Boston
`Irene Rubinson, Advertising Manager,
`Raven Press, New York
`
`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. Subscripti'on rates for Vol. I, 198112: 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.
`
`Sanofi/Regeneron Ex. 1 004, pg 63
`
`Merck Ex. 1004, pg 63
`
`

`
`J1111rllrrl o( .\fo/,·clllm r/11.1 At•fllinl (i,·w·rir 1
`(:_1!7- J-11 ,,., I'>'X! fl.avcn l'rc". N~w y,,k
`
`Transformation of Mammalian Cells to
`Antibiotic Resistance with a Bacterial Gene Under
`Control of the SV 40 Early Region Promoter
`
`P. J. Southern and P. Berg
`
`lkpartnrclll ~~f' Biochcmi.1·tn·, Sttll({ord Unil'ersit_\' Medic11f Center, Sttlll]rml, Col({omi11 , U.S.A.
`
`Summary: A bacterial gene (nco) conferring resistance to neomycin(cid:173)
`kanamycin antibiotics has been inserted into SV40 hybriu plasmiu
`vectors and introduced into c.ultureu mammalian cells by DNA trans(cid:173)
`fcction. Whereas normal cells are killed by the antibiotic G418, those
`that acquire anu express neo continue to grow in the presence of G418.
`In the course of the 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(cid:173)
`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 (I) or be(cid:173)
`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 in1l1 recipient cells by either
`the calcium phosphate precipitation technique
`
`Received January 4. 1982: accepted February 18, 1982.
`Adurcss correspondence and reprint requests to Dr.
`P. J. Southern. Dcp;lflmcnl uf Immunopathology,
`~ripps Clinic and Rescan.:h Foundation, La Julia, CA
`9!037.
`
`(11), DEAE-dextran (12), or microtnjection
`(13, 14). The pioneering experiments for this
`approach relied on the transformation of TK(cid:173)
`mammalian cell lines to a TK+ phenotype
`with the herpes simplex thymidine kinase gene
`(15 -17). Subsequently, unrelated DNA se(cid:173)
`quences have been integrated with the thy(cid:173)
`midine kinase gene in either linked or co(cid:173)
`transformation experiments ( 18, 19). Transfor(cid:173)
`mation of appropriate mutant cell lines has also
`been demonstrated with cellular DNA se(cid:173)
`quences apd this approach has allowed the
`isolation of the chicken thymidine kinase gene
`(20) and the hamster adenine phosphoribosyl
`transferase gene (21). Morphologic transfor(cid:173)
`mation of normal cells forms the basis of cur(cid:173)
`rent attempts to isolate cellular oncogenes
`(22.23).
`Unfortunately, experiments that rely upon
`complementation of cell mutations by trans-
`
`'
`
`327
`
`Sanofi/Regeneron Ex. 1 004, pg 64
`
`Merck Ex. 1004, pg 64
`
`

`
`328
`
`P . .1. sourur.R.v :IMJ 1'. at~RG
`
`duced genes are limited by the availability of
`mutant mammalian cell types to serve as gene
`rel:ipients. 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-resistant transformants after
`tr:~nsfection nf normal cells with ON A rrom
`drug-resistant cells (24) exemplifies this ap(cid:173)
`proach. However, transformation for metho(cid:173)
`trexate-resistant dihydrofolate reductase is in(cid:173)
`frequent and, therefore, the utility for cotrans(cid:173)
`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 (I 0), and E. coli
`xanthine-guanine phosphoribosyl transferase
`(8) can induce the synthesis of the corre(cid:173)
`sponding gene products in mammalian cells.
`The expression of the bacterial gene, gpt, per(cid:173)
`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).
`In this paper, we describe a second bacterial
`ger\e 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(cid:173)
`lian cells relies on cell killing by an amino(cid:173)
`glycoside antibiotic, G418 (27). The structure of
`G418 resembles gentamicin, neomycin, and
`kanamycin (28) but, unlike these related com(cid:173)
`pounds, G418 interferes with the function of
`80S ribosomes and blocks protein synthesis in
`eukaryotic cells (27). These aminoglycoside
`antibiotics can be inactivated by the bacterial
`phosphotransferascs, APH(3')11 and APH(3')1
`encoded by transposons Tn5 and Tn601, re(cid:173)
`spectively (29). Jiminez and Davies (30)
`showed that yeast could be genetically trans(cid:173)
`formed to G418 resistance by the phospho(cid:173)
`transferase gene contained in Tn60 1 DNA.
`Thus, it sec:med feasible (suggested by Sydney
`
`J. Mol. A/>!•1. (it'IWI .. Vol. I. N11. 4. /'1112
`
`Brenner) that the acquisition ami expre~ion 0
`the phosphotransferasc gene by mammaliar
`cells might confer resistance to G41 g to\icity
`Accordingly, the" phosphotransfcrase gene
`from Tn5 (designated here nco) (29,31) was in·
`troduced into the mammalian transcripti-on unil
`of the pSV plasmid vectors. In this arrange·
`mcnt the SY40 early promoter is 5'-proximal.
`and an intron and plllyac..lenylation signal arc
`3'-proximal. to the neogene (8,25). Transfcc·
`tion of a wide variety of mammalian cell line!
`with these pSV-neo recombinants yields stable
`t ransformants that are resistant to G~ 18 at a
`frequency of one transformant per 10~-10~
`transfected cells. Our do.~ta suggest that trans·
`formation results from the acquisition. m:lintc·
`nance, and continued expression of the neo
`gene in the cellular genome. Concurrent with
`our experiments. Colbere-Garapin el al. (3:!)
`achieved mammalian cell transformation to
`G418 resistance with recombinant DNA con(cid:173)
`taining the Tn5 nco gene linked to the herpes
`thymidine kinase promoter DNA segment.
`
`MATERIALS AND METHODS
`
`Cells
`
`A wide range of cultured mammalian celb
`are sensitive to the antibiotic G41S tT01ble II
`and several of these have been used for trans(cid:173)
`formation experiments. The cells were ruu·
`Linely maintained in Dulbecco-modif~ed Eagle
`medium containing 10% newborn calf serum.
`penicillin and streptomycin, and the indicated
`concentrations of G418 were ac..lded to the
`medium.
`
`Enzymes
`
`Restriction enzymes were purcha~d front
`New England Biolabs and Bethesda Rescun;h
`Laboratories and digestions were perrorm.:J
`according to the surplier's specilicatiuns. 'fJ
`polynucleotide kinase was purchaseJ front
`New England Nudcar and S I nuclease front
`Boehringer Mannhcim. T4 DNA ligase ami f.
`coli DNA polymerase 1 were kindly pnwiJ~J
`hy S. Scherer, Stanford University.
`
`Sanofi/Regeneron Ex. 1 004, pg 65
`
`Merck Ex. 1004, pg 65
`
`

`
`Antibiotic G418
`
`Samples of antibiotic G41N were generously
`rrovidcu by Dr. P. J. L. Daniels of Schcring
`corporation. Stock solutions containing 4
`mg}ml G4Hl in 100 mM N-2-hyuroxycthylpi(cid:173)
`raazine-N'-2'-cthancsulphonic acid buffer. pH
`7.3. were stored at - 20°C and au lieu in ap(cid:173)
`propriate amounts to the cell culture meuium.
`rhe G418 concentration refers to the actual
`amount of drug in the solution anu takes into
`account that the solid material was only 40-
`501/c G418.
`
`DNA Transfection and Selection of
`Transformed Cells
`
`Supercoiled plasmid DNA, without added
`:arrier DNA. was introduced into tissue cul(cid:173)
`ure cells (10 P,g for approximately 5 x 10 11
`:t:lls) using the calcium phosphate precipitation
`echnique (II) with the addition of a glycerol
`;hock after 4 h (33). About 48 h after exposure
`o DNA, the cells were trypsinized and re(cid:173)
`>lated at a I :20 dilution. Within 12-16 h, G418
`~·as added to the medium at a concentration of
`~00 p,glml. The medium plus drug was changed
`:very 4 to 5 days. Colonies were first detected
`1fter about 7 days in the selective medium and,
`r-13 days later, independent colonies were
`rypsinized in cloning cylinders and transferred
`o microtiter wells. When the colonies were
`.mall, the transplanted cells were grown non(cid:173)
`electively for an initial 2-3 days. Once estab(cid:173)
`ishcd, the clones were expanded to stable cell
`ines in medium containing 400 ,u.glml G418. In
`;omc instances, the initial selection and sub(cid:173)
`:loning used 400 p,g/ml of G418 but the trans(cid:173)
`\lrmed cells were subsequently maintained in
`~00 p.g/ml of G418.
`The selection strategy of permitting cell
`:rowth prior to the addition of G418 was
`1dopteJ because a significant reduction in the
`ransformation frequency occurred if G418 was
`1dded before 48 h. The tra~sfected cells were
`eplated at lower cell density because G418 is
`nost effective against dividing cells. Con(cid:173)
`cquently. if cells become stably transformed
`
`early _after transfc~.:tion, cell division prior to
`selection may result in overestimation of the
`transformation frequency. Nevertheless, none
`of the transformants from randomly selected
`colonies appeareJ to have the same organiza(cid:173)
`tion or the integrated pSV-neo DNA.
`
`Analysis of Transformed Cell DNAs
`for pSV-neo Sequences
`High molecular weight cellular DNA was
`~xtracted as .described by Wigler et al. (17),
`mcubated With an excess of restriction en(cid:173)
`zyme, and the digests were separated by elec(cid:173)
`trophoresis in 0.8% agarose gels. After a mild
`depu ri nation reaction (34), the DNA was
`transferred from the gel to diazobenzyloxy(cid:173)
`methyl paper (DBM paper) (35), hybridized
`with ra~ioactively labeled DNA probes (36),
`and rad10autographed 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
`'semicontluent cultures of transformed cells as
`described previously (38). The RNA was sepa(cid:173)
`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(cid:173)
`tion fragments at their 5'-ends with [y-a2 P](cid:173)
`adenosine triphosphate and polynucleotide
`kinase ( 41 ). The DNA probes were hybridized
`with RNA samples under conditions of DNA
`excess, RNA-DNA hybrids were digested with
`Sl nuclease, and the protected fragments were
`analyzed by gel electrophoresis (39,40).
`
`Protein Labeling and
`lmmunoprecipitation Reactions
`Semicontluent plates of pSV2-neo trans(cid:173)
`formed cell lines were labeled for 14 h at 37°C
`
`J. Mol. Appl. (,',·rl<'l., \'of. I, 1\'o . .J, 1982
`
`Sanofi/Regeneron Ex. 1 004, pg 66
`
`Merck Ex. 1004, pg 66
`
`

`
`330
`
`P. J. SOUTIII:'RN ANJ) !'. IJJ:RG
`
`with [aH]Ieucine (200 !J.C'i/plate, specific activ(cid:173)
`ity 55 Cilmmol, New England Nuclear Labo(cid:173)
`ratory). The soluble proteins were extracted
`from approximately 2 x 10; cells (7) and im(cid:173)
`munoprccipitated with an antiphosphotransfer(cid:173)
`ase APH(3')ll serum (provided by J. Davies,
`Geneva). EschNichia ('()/i cells (HB 101) con(cid:173)
`taining plasmids were grown to approximately
`2 x wx cells/ml in M9 minimal medium plus
`glucose with supplements of leucine, proline,
`threonine, and thiamine. Samples of the cultures
`(0.5 ml) were washed and resuspended in the
`same medium lacking leucine and then r:'H]leu(cid:173)
`cine was added (200 JA.Cilml) 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(cid:173)
`trifugation at l4,000g for 10 min and the super(cid:173)
`natant was used directly for immunoprecipitation
`reactions. After incubation overnight at 0°C, the
`immune complexes were adsorbed to inacti(cid:173)
`vated S. aureus cells (lgGsorb, Enzyme Cen(cid:173)
`ter, Boston) and removed by centrifugation
`(42). The S. aureus cells were washed exten(cid:173)
`sively and the bound proteins were eluted and
`electrophoresed in SDS polyacrylamide gels
`(43). After electrophoresis, the gels were
`treated with EN:'HANCE (New England Nu(cid:173)
`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 microliter wells in a medium
`supplemented with various concentrations of
`G418. Even at the highest drug concentration
`tested (800 !J.g/ml), sensitive cells divided once
`or twice before cytotoxicity was ohservcd. The
`response time for cell killing appears to corre(cid:173)
`late with growth rate, since the most rapidly
`growing cells arc killed in the shortest inter(cid:173)
`vals .. At lower concentrations of G418 (I 00
`!J.g/ml) there is a significant delay but the cells
`arc killed eventually. All of the cell lines that
`
`J. Mol. ,\l'r•l. (;,.,..,., Vol. I. No. -1. IW12
`
`TAULE I. G--1/il·.l<'llxitil·,· 111<11111/llliiull cdllinu"
`
`Mouse
`
`Mon~cy
`
`<C'V I
`CVI·P
`TC7
`cos
`
`L
`Lt~
`3T3
`JTtl
`PCC4
`F9
`MEL
`
`Human
`
`I.NSV
`He La
`K·56Z
`
`"So f<Lr, no ..:ell line has been rounc.l that is na!Urallv
`resistant lo G4il!.
`·
`
`have been tested (Table I) are killed by G418
`but CV I and Hela are unusual because. at
`high cell density, these cells may require 10-14
`days in G418 (400 !J.g/ml) before the cell killing
`can be observed.
`
`Construction of Recombinant
`PJasmids Containing neo
`
`The bacterial transposon Tn5 encodes a gene
`(neo) whose protein product-a phosphotrans(cid:173)
`ferase (APH(3')11)--confers resistance to the
`kanamycin-neomycin group of antibiotics (311.
`From the studies of the organization of Tn5
`DNA, Reznikoff and colleagues (44.45) were
`able to identify the DNA segment that is essen(cid:173)
`tial for the expression of neo. The Co IE I :Tn5
`plasmid pRZI12 (44), a deleted form of the or(cid:173)
`gina! CoJEI:Tn5 hybrid plasmid (pRZ102. Fig.
`1), was the source of the neo DNA segmenl.
`pRZJI2 DNA was digested to completion with
`Hincll endonuclease and the 2.5 kb neo DN.-\
`segment was obtained by agarose gel electro(cid:173)
`phoresis. After ligafing a dc~:anuclcotide se·
`quence containing the BamHl restriction site
`(Collaborative Research) to the ends of the nl!O
`segment (46) the mixture was digested with an
`excess of BamHI and l/i11d11l restriction en·
`donucleases and the resulting 1.4 kb nco DNA
`fragment was puritied by gel clectrophore~i~.
`This fragment. containing /linJ111 and BwnHI
`cohesive ends at the 5'- and 3'-ends, resp~~·
`tivcly, was inserted between the //indlll anJ
`Ba111Hl restrktion sites in pBR322 DNA. Tlu:
`resulting plasmid, pBR-nco (foig. 1), confers r~·
`sistancc in /~·. coli to hoth ampidllin anJ
`
`Sanofi/Regeneron Ex. 1 004, pg 67
`
`Merck Ex. 1004, pg 67
`
`

`
`'I RANSFORMA TJON TO ANrtEJIOT/C RES/SJANC/~·
`
`33/
`
`H~~~~
`
`Hmr/111
`
`1 Brill
`
`s.-:'1
`•
`:ATG
`'
`'
`,.,..... ne-0-...1
`
`Hnu/111
`Bqi:IJ
`
`pRZ102
`
`H111cll
`I
`
`{Honclll Hmdlll
`HJio/1
`[BI}JII
`I
`I
`~
`:ATS
`,.....,.. neo __..,
`+ Hincll CLEAVAGE
`
`pR21l2
`
`Hmcll
`
`Hmr/111
`IBglll Hincll
`
`Hmdlll ! BamHI LINKERS
`
`B.u11HI • Hin{/111 CLEAVAGE
`ISOLATION OF neoGENE FRAGMENT
`
`jli-1111
`B.1mHI
`~w..d
`
`'INSERTION INTO pBR322
`~SELECTION FOR AmpR,NeoR
`
`Rl
`Him/Ill
`AmeR
`lr Bglll
`
`pBR322
`on
`
`pBRni!O
`
`Eco-neo
`
`BamHJ
`
`'IG. 1. Organizalion oltransposon Tn5 and scheme for the
`onstruction of recombinant plasmid pBR-neo. The plasmid
`1RZ1 02 contains a complete copy of Tn5 inserted into ColE 1.
`'he inverted repeat sequences at the ends ot the transposon
`reshown as thick lines. The region essential for expression
`•I neomycin resistance in E. coli (neo) is indicated together
`1ith the ATG codon that initiates the coding sequence ol the
`•hosphotranslerase. Relevanl restriction endonuclease rec(cid:173)
`•gnllion sites are included in the diagram. pRZ112 was de(cid:173)
`lved from pRZ102 by partial digestion with Hincll endonu(cid:173)
`lease and ligation to eliminate a large segment of Tn5 DNA to
`,e 3' side of neo (Hincll endonuclease cleaves at Sa/1 and
`'pal endonuclease recognition sites.) Details of the ma(cid:173)
`ipulations involved with the construction of p8R-neo are
`1iven in the text.
`
`1eomycin: since the neo DNA segment inter(cid:173)
`upts the tetracycline resistance ·gene, cells
`<~rrying this plasmid arc sensitive to this anti(cid:173)
`tiotic.
`The cloned neo DNA segment was readily
`ntruduced into the plasmid vector-pSV2 (8)
`Fig. 2) by excising the ,8-globin eDNA segment
`rom pSV2-.BG (25) with Hindiii and BRill en-
`
`llonucleasc digcslion and substituting the neo
`fragment viu the corresponding cohesive ends.
`The pSV3-neo and pSV 5-nco (Fig. 2) deriva(cid:173)
`tives were constructed from pSV2-neo as pre(cid:173)
`viously described (8.25). Each of the pSV-neo
`plasmids replicates efficiently in E. coli strain
`H B I 0 I and confers resistance to ampicillin and
`neomycin. Cloned isolates of each of the re(cid:173)
`combinant plasmids were shown to have the
`anticipated structures by appropriate restric(cid:173)
`tion enzyme analyses (data not shown).
`
`Cell Transformation with Recombinant
`Plasmids Containing neo
`
`Transfections of Ltk- cells with pRZI12 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(cid:173)
`formants have been recovered with pRZII2
`DNA and only one transformant has been iso(cid:173)
`lated after transfection with pBR-neo (fre(cid:173)
`quency about one transformant in 10' trans(cid:173)
`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 10 7
`transfected cells).
`In contrast to the low frequencies of G418-
`resistant transformation with pRZII2 (ColE 1-
`neo) or pBR-neo plasmid DNAs, several dif(cid:173)
`ferent mammalian cell lines were transformed
`to G418 resistance at relatively high frequency
`with the pSV-neo plasmid derivatives (about
`one transformant in 10 4 to to··· transfected
`cells). Although the transformation frequencies
`with the different pSV-neo recombinants fall
`within~ relatively narrow range,- there is a con(cid:173)
`sistent small difference which reflects the
`plasmid's potential for replication in different
`host cells (Table 2). For example, the fre(cid:173)
`quency of stable G418-resistant monkey cell
`transformants is two- to three-fold lower with
`pSV3-neo than with pSV5-nco, whereas the
`converse applies to the formation of G418-
`rcsistant mouse cell transformants. This differ-
`
`J . .\1,>/. A!•t•l. <ic'lll'l .. \'of. J. No.-1. f'JX]
`
`Sanofi/Regeneron Ex. 1 004, pg 68
`
`Merck Ex. 1004, pg 68
`
`

`
`pBR322 Orl
`
`Pv11ll
`
`neo
`
`p8R322 ori
`
`Pvull
`
`/
`
`,.
`
`/
`
`,. '
`
`/ ' ~'"
`
`SV40- Tag
`
`p8A322 0r1
`
`Pv<JII
`
`/
`
`'
`'
`' \
`
`\ ' \
`
`\
`
`'
`
`//'))'"
`,\ I
`')r
`
`·
`
`PY-
`
`orr
`
`neo
`neo
`FIG . .2. Structures of the pSV-neo hybrid plasrnids The pSV plasmids are composed of DNA segments from various source
`p8R322 DNA, represented by the solid black arc. contarn:; the p8R322 origin of DNA replication (p8R322 ori) and the J;l-lactama
`gene (Amp" I, tne hate ned segmenlrepresenrs the neogene (1.4 kb fragment, Fig. 1); SV40 DNA sequences are indicated as lr
`dolled segments. The SV40 origrn of DNA rephcatron (SV40 on) and the SV40 early promoter are present on a small fragme
`(SV40 map umts 0.71-0.65) 1mmedrately 5' to the neo segmenL The plasm ids pSV3-neo and pSVS-neo were constructed fro
`pSV2·neo by rnsertion ol mtner an rntact SV40 early region or an intact polyoma early region that contains a duplication of 11
`viral orrgrn of rcplicatoon (8).
`
`ence 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
`tmnsformations with pSV3-gpt and pSV5-gpt
`recombinants (26).
`
`Stable Integration of neo DNA Sequence!
`after Transfection into Cultured Cells
`
`The content and organization of neo ON;
`sequences have been examined in a number o
`stable G418 transformed cell lines. The Ltk- o
`
`TABLE 2. Tran.,.fi>r~nuriml .fi·ctflll'llci<'.l' }in· l'l'l'om/Jimur/
`1/('(1 plll.\lllid.l ,,
`
`Hu~t cells
`
`Ltk
`
`3T6
`
`TC7
`
`pSV2-nco
`pSVJ-nco
`pSV5-ncu
`pUR-nco
`
`-]X 10 I
`.. 3 X 10 '
`-)X 10
`.5 X 10 ,,
`
`I
`
`I
`
`7 X 10
`1.5 X 10
`4 X Ill
`I X 10
`
`)( 10'
`6 X 10
`2 X
`IU '
`Nn ~nklllic~ i~ubrcd
`
`"Transl'ormaliun frcyucncy is expressed as I he fraction of cells plated
`lh:at produce viable cni<Hlic~ in sclc~livc mclliurn fl•llnwing transfccti,m
`wilh satur;uing lo:vcls ,,f DNA.
`
`J . . 1/.,1. :11'1•1. c;,.,,..,., l'o/. /,No . .J. 19111
`
`Sanofi/Regeneron Ex. 1 004, pg 69
`
`Merck Ex. 1004, pg 69
`
`

`
`TUrlNSFORMA liON 7'0 ANTIIJJOT/C R.ESISTANC'J:'
`
`333
`
`H6 transformants arising from tmnsfcctions
`~1·ith pSV2-neo, pSV3-neo. or pSV5-neo do not
`contain plasmid-related se4ucnces (limits of
`detection 0.5 plasmid molecules per cell} in the
`loW molecular weight DNA of a Hirt superna(cid:173)
`tant (47). However, cell DNA obtained from
`G41 !!-resistant transformants contained nucle(cid:173)
`otide sequences homologous to the transfecting
`plasmid DNA. High molecular weight DNA
`from a reprt!sentative set of pSV2-neo Ltk(cid:173)
`transformants was cleaved with EcoRI restric(cid:173)
`tion endonuclease, electrophoresed in an
`agarose gel. transferred to DBM paper. and
`then hybridized with radioactively labeled
`pSV2-neo DNA (Fig. 3). Since pSV2-rieo DNA
`contains a single EcoRI restriction site, each
`integrated plasmid copy should produce two
`bands after hybridization with the pSV2-neo
`probe if there is no rearrangement or scram(cid:173)
`bling of the plasmid DNA sequences during in(cid:173)
`tegration. Most of the transformants that have
`~een examined appear to have a low plasmid
`copy number, generally one to five copies per
`cell. Transformant 20 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 EcoR1
`endonuclease (Fig. 3). Since no circular plas(cid:173)
`mid DNA was present in the Hirt supernatant
`fraction prepared from 20 cells and the high
`molecular weight DNA of the Hirt pellet hy(cid:173)
`bridized strongly to a radioactively labeled
`pSV2-neo probe (results not shown), it is likely
`thattransformant 2G,contains either a tandem
`array of intcgratt!d plasmid DNA or autono(cid:173)
`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(cid:173)
`quences do not change after passaging in non(cid:173)
`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(cid:173)
`taining G418 (200 J..Lg/ml); such cells remain
`fully resistant to 0418 when challenged 111
`
`~---"----,
`I 2F 1 2G I 2J I 2K I M
`
`I
`
`20 kb-
`
`5.6 kb-
`
`l •
`
`LINEAR
`pSV2neo
`
`1 kb-
`
`FIG. 3. Detection of pSV2-neo DNA sequences in trans(cid:173)
`formed cells. High molecular weight cell DNA was extracted
`from representative G418-resistant Ltk" cell lines that had
`been transformed with pSV2-neo plasmid DNA. The DNA
`samples were digested with an excess of EcoRI restriction
`endonuclease and then fractionated by electrophoresis in a
`0.6% agarose gel. DNA In the gel was transferred to OBM
`paper a'nd hybridized with radioactively labeled pSV2·neo
`DNA. There is a single recognition site in pSV2-neo DNA lor
`EcoRI cleavage and the marker track (M) shows the position
`of pSV2-neo linear DNA. Approximate molecular sizes were
`derived from A DNA restriction fragments in an adjacent slot
`on the gel.
`
`selective medium. The relative plating effi(cid:173)
`ciency oft he 2F or 20 cell lines after seeding in
`the presence or absence of G418 is within ex(cid:173)
`perimental variation, the same (ratio 0.6 to
`0.9: I .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
`at different times during their growth in the
`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. ,\fol. -'1'1'1. G<'ll<'/., Vol. J. No. 4, IV.~]
`
`Sanofi/Regeneron Ex. 1 004, pg 70
`
`Merck Ex. 1004, pg 70
`
`

`
`33-1
`
`P. J. SOVTIIERN ANO P. Bf:RG
`
`Ltk.-pSV2nec2G
`Llk- pSVJ1neo2G
`Ltk- pSV2neo2F
`~ ~ ~
`'"'
`,'b ~
`,'b
`,'b
`/
`()
`c)'
`c)'
`~"" ~ &
`<:}'
`f)
`I "
`I
`');
`I •
`I "
`'
`I X
`I "
`I
`
`'"'
`
`'\,()
`I
`
`I ~ I
`
`X
`
`I
`
`•
`
`FIG. 4. Detection of pSV2-neo DNA se(cid:173)
`quences alter extended passaging of
`transformed cells. High molecular weight
`chromosomal DNA was extracted from
`parallel cultures of the transformed cell
`lines 2F and 2G that had been maintained
`in the presence (+G418) or absence
`(-G418) of G418 (see teKt}. DNA samples
`were digested with EcoRI endonuclease
`and analyzed as described in Fig. 3. The
`tracks at the extremities of the gel
`(labeled 2F and 2G} contain samples of
`the original DNA preparations (see Fig. 3).
`The central track contains parental Ltk"
`cell DNA. The panel at the right of the fig(cid:173)
`ure is a shorter exposure during au(cid:173)
`toradiography of the Ltk· pSV2-neo 2G
`cell DNA.
`
`-
`
`-
`
`_LINEA
`pSV2nE
`
`-·.
`
`•
`
`ble during growth of these two transformed cell
`lines under selective or nonselective condi(cid:173)
`tions.
`We sought to determine if stable transforma(cid:173)
`tion for the transduced marker could occur in
`the absence of selection. Acco.rdingly, semi(cid:173)
`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(cid:173)
`other set was propagated in the absence of
`0418 for about 12 generations (2 weeks) and
`then the G418 selection was applied. The
`transformation frequency in the first set was
`10-" to JQ- 5 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(cid:173)
`formed and normal cells divide at the same rate
`in medium lacking G418 and considering the
`inherent inaccuracies of the experiment, it
`
`seems that the neo marker is associated wit!
`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-resistanl
`transformants has been confirmed by the de·
`tection of neo mRNAs and phosphotransfcrase
`protein. Cytoplasmic, polyiA) .. RNA was iso·
`Ia ted from pSV2-neo transformed Llk · cells
`(isolates F and G) and hybridized to an end·
`labeled ON A probe specific for pSV:!-neo se·
`quenccs. In each sample, digestion with Sl
`nuclease and clectrorhorcsis of the protected
`DNA in an agarose gcJ (39,40) yielded a single
`fragment of 1500 bases (Fig. 5). The prc.xfuction
`of a 1500 base fragment is consistent with the
`occurrence of an RNA that ex tends throughout
`the length of the nco segment and suggesls I hat
`
`J. Mol. Aflfl/. (irnrt .. Vol./, No.4, /9X2
`
`Sanofi/Regeneron Ex. 1 004, pg 71
`
`Merck Ex. 1004, pg 71
`
`

`
`I
`~
`...1
`
`u.
`N
`
`l'l
`N
`
`'ti
`~ .!;: ~
`l:
`
`-
`
`-PROBE
`
`·--
`
`bp
`-1500
`-1430
`
`-1110
`
`FIG. 5. Delection of neo sequences 1n cyiO·
`plasmic RNA extracted from pSV2·neo trans(cid:173)
`formed Ltk · cells. A DNA probe was prepared by
`5' end-labeling with "Pat the Bam HI restrict1on
`site at the 3' extrem1ty of the neo DNA fragment.
`(The Bam HI site was not preserved in the con(cid:173)
`struction of pSV2-neo and the probe lor this ex(cid:173)
`periment was prepared lrcim another derivative
`plasmid.) Rele~ant restriction endonuclease
`cleavage sites are indicated in the diagram.
`RNA-DNA hybrids were formed under condi(cid:173)
`tions of DNA excess (40) and the S1 nuclease
`digests were fractionated on a 1% neutral
`agarose gel Marker bands were produced by
`d1gesting the DNA probe with Bg/1. Hlndlll and
`Bglll restriction endonucleases. The DNA trag·
`men! protected by the transformed cell RNAs is
`shown in the diagram as a continuous line
`under the representation of the DNA probe.
`
`PROBE FOR pSV