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`WOLECULAR
`AND APPLIED
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`/OLUIVIP P1-NUMBER Z1-198*?-PAVEN PPESS
`
`Sanofi/Regeneron 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 021 I4, U.S.A.
`
`Editorial Board
`
`John Abelson, La Jolla
`Frederick Ausubel, Cambridge
`John R. Bedbrook, Canberra
`Lawrence Bogorad, Cambridge
`Mary-Dell Chilton, St. Louis
`Richard A. Flavell, London
`Raymond F. Gesteland, Salt Lake Ciry
`Gary Gussin. Iowa City
`Dean H. Hamer, Bethesda
`Masayorl lnouye, Stony Brook
`Robert Kamen. London
`Yuet Wai Kan, Sun FI‘(lIl('l.\‘(‘I)
`Laurence H. Kedes, Palo Alto
`Daniel Klessig, Salt Lake Cit)‘
`Thomas Maniatis, Cambridge
`David W. Martin, Jr., San Francisco
`Marc Van Montagu, Gem
`Richard C. Mulligan, Cambridge
`Alexander Rich, Cambridge
`Jcff Schell, Cologne
`James Shepard, Manhattan
`John Shine, Canberra
`Juan A. Steitz, New Haven
`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, Assistant
`Editor-in-Chief, Boston
`Irene Rublnson, Advertising Manager.
`Raven Press, New York
`
`to the
`
`Volume 1, Number 4, 1982 Blomodlul Library UCSD 1
`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 Marylane 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 3 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, 198!/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.
`
`Sanofi/Regeneron Ex. 1004, pg 63
`
`

`
`_[,mrm1f 1’_fA\'UIl'('I1IllI‘ uml /lppliml U('Ilt'Ilt .\
`1327-3-1|
`'¢'v I951 Raven |’res~.. New York
`
`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
`
`l)¢'purrm('nI ufBim'/twni.rlr\', 5mn_/}u'¢/ UIll|'('I‘.Vll_\' Mc'di('u/ Ct‘I1l(’I', Smufnrc/. Ct!/fforiiiu. U.S.A.
`
`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 ofG4l8 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 ofDNA
`and to propagate the recombinants with the aid
`of helper viruses in cultured animal cells
`13- 10). Alternatively. exogenous DNA may be
`introduced directly into recipient cells by either
`the calcium phosphate precipitation technique
`
`Receivedjanuary 4. 1982'. accepted February 18. 1982.
`Address correspondence and reprint requests to Dr.
`P.J. Southern. Department of lmmunopathology.
`gsfflpps Clinic and Research Foundation, La Julla. CA
`.037.
`
`327
`
`(ll), 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-
`
`Sanofi/Regeneron Ex. 1004, pg 64
`
`

`
`328
`
`P. .I.
`
`.S'()U'I‘HI-IRN AN!) I’. Hl:’R(}
`
`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 It
`solution to this difficulty. The isolation of
`methotrexate—resistant
`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 B-globin (25),
`mouse dihydrofolate reductase (10), and E. colt’
`xanthine-guanine phosphoribosyl
`transferase
`(8) can induce the synthesis of the corre-
`sponding gene products in mammalian cells.
`The expression of the 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).
`In 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, 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
`phosphotransferases, APH(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-
`transferase gene contained in Tn6(ll DNA.
`Thus. it seemed feasible (suggested by Sydney
`
`J. Mal. A/up/. (r'l'H4'I., Val. /. No. 4. I982
`
`Brenner) that the acquisition and expression 0
`the phosphotransferase gene by mammaliur
`cells might conferbresistanee to G4|8 toxicity
`Accordingly,
`the phosphotransferase gene
`from Tn5 (designated here neo) (29.3!) was in-
`troduced into the mammalian transcription unit
`of the pSV plasmid vectors.
`In this arrange-
`ment the SV40 early promoter is 5'-proximal.
`and an intron and polyadenylation signal are
`3'-proximal.
`to the neo gene (8.25). Transfec-
`tion of a wide variety of mammalian cell 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. Colbere-Garapin et al.
`(32)
`achieved mammalian cell
`transformation to
`0418 resistance with recombinant DNA con-
`
`taining the Tn5 neo gene linked to the herpes
`thymidine kinase promoter DNA segment.
`
`' MATERIALS AND METHODS
`
`Cells
`
`A wide range of cultured mammalian cells
`are sensitive to the antibiotic G4l8 (Table ll
`and several of these have been used for trans-
`
`formation experiments. The cells were rou-
`tinely maintained in Dulbeeco—modified Eagle
`medium containing I0”/c. newborn calf serum.
`penicillin and streptomycin. and the indicated
`concentrations of G418 were added to the
`medium.
`
`Enzymes
`
`Restriction enzymes were purchased from
`New England Biolabs and Bethesda Research
`Laboratories and digestions were perform!-‘*5
`according to the supplier's specifications. T4
`polynucleotide kinase was purchased frat“
`New England Nuclear and SI nuclease frttll‘
`Boehringer Mannheim. T4 DNA ligase and E-
`mil DNA riolymera.~:e l were kindly provided
`by S. Schercr. Stanford University.
`
`Sanofi/Regeneron Ex. 1004, pg 65
`
`

`
`'mA,v.s'I-‘om/,4 7/()/V 'I() zlNTIBl()'I'I(' R[;'SI.S"l'/tNCE
`
`329
`
`Antibiotic G4l8
`
`Samples of antibiotic G418 were generously
`provided by Dr. P. J. L. Dtiniels of Schering
`corporation. Stock solutions containing 4
`mg/ml G4l8 in I00 mM N-2-hydroxyethylpi-
`pcrazine—N'-2’—ethanesulphonic acid buffer, pH
`7_3, were stored at —20°C and added in ap-
`proprldte 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-
`50'’)? G4l8.
`
`DNA Transfection and Selection of
`Transformed Cells
`
`Supercoiled plasmid DNA, without added
`:urrier DNA. was introduced into tissue cul-
`ure cells (10 itg for approximately 5 x I0"
`:ell5) using the calcium phosphate precipitation
`echnique (11) with the addition of a glycerol
`;hock 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
`
`l00 p.g/ml. The medium plus drug was changed
`:very 4 to 5 days. Colonies were first detected
`ifler about 7 days in the selective medium and,
`7-13 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. ln
`.onie instances,
`the initial selection and sub-
`loning used 400 pg/ml of G4l8 but the trans-
`ormed cells were subsequently maintained in
`100 pg/ml of G4l8.
`The selection strategy of permitting cell
`zrowth prior to the addition of G418 was
`idopted because 21 significant reduction in the
`ransformation frequency occurred if G418 was
`l(.l(.lCLl before 48 h. The trarisfected cells were
`eplated at lower cell density because G418 is
`nost effective against dividing cells. Con-
`cquently.
`if cells become stably transformed
`
`early after transfection. 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. (I7),
`incubated with an excess of restriction en-
`
`zyme, and the digests were separated by elec-
`trophoresis 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 [)«-‘'”P]—
`adenosine triphosphate and polynueleotide
`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 I4 h at 37°C
`
`J, Ah;/_ App’. (l‘l.'IIl‘f.,
`
`l'u/. I. No. 4, 1982
`
`Sanofi/Regeneron Ex. 1004, pg 66
`
`

`
`330
`
`P. J. .S'()U'I'llI-{RN AND I’. I3!-ERG
`
`with ["H]leucine (200 ;.LCi/plate. specific activ-
`ity 55 Ci/mmol. New England Nuclear Labo-
`ratory). The soluble proteins were extracted
`from approximately 2 X I0’ cells (7) and im-
`munoprecipitated with an antiphosphotransfer-
`ase APH(3’)ll serum (provided by J. Davies.
`Geneva). Est-/tcricliia (‘vii cells (HBl0l) con-
`taining plasmids were grown to approximately
`2 X l0" 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 Ieucine and then [“l-l]|eu-
`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. tmrcns cells (IgGsorb, Enzyme Cen-
`ter. Boston) and removed by centrifugation
`(42). The S. aureus 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“I-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 pg/ml), 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)t)
`pg/ml) there is a significant delay but the cells
`are killed eventually. All of the cell lines that
`
`J. Mul. Appl. (;1'll¢‘!.. Vol. I, NH. 4. I932
`
`TABLE I. G-lI8—.t"-/I.\’iIit':- Inu/mm:/i'uu well limx"
`
`Monkey
`
`(‘V1
`CVI-P
`'l‘C7
`COS
`
`Human
`
`LNSV
`He La
`K-56'.’
`
`Mouse
`
`L
`Ltk
`31'}
`1T6
`PCC4
`F9
`MEL
`
`" So far. no cell
`resistant to G-H8.
`
`line has been Found that is naturttlly
`
`have been tested (Table l) are killed by G418
`but CVI and HeLa are unusual because. at
`
`high cell density, these cells may require l0— l4
`days in G418 (400 pg/ml) 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 (API-l(3')ll)—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 ColEl:Tn5
`plasmid pRZl I2 (44). a deleted form of the or-
`ginal Col El:Tn5 hybrid plasmid (pRZl02. Fig-
`l). was the source of the neo DNA segmen|-
`pRZl I2 DNA was digested to completion with
`Hincll endonuclease and the 2.5 kb neo DNA
`
`segment was obtained by agarose gel electro-
`phoresis. After ligating a deeanucleotide se-
`quence containing the BmnHl restriction silt‘
`(Collaborative Research) to the ends ofthe n60
`
`segment (46) the mixture was digested with an
`excess of BumHl and Ilindlll restriction 8"’
`donucleases and the resulting I.4 kb neo DNA
`fragment was purilied by gel e|cClI'0ph0resi.\-
`This fragment. containing Ilindlll and BumHl
`cohesive ends at the 5’- and 3'-ends, re$P¢"'
`tively. was inserted between the Hindlll and
`BmnHl restriction sites in pBR322 DNA. Tl“
`resulting plasmid. pBR-neo (Fig. I). confers re-
`sistance in If.
`(‘U/l
`to both umpicillin and
`
`Sanofi/Regeneron Ex. 1004, pg 67
`
`

`
`Hm NS]-'()R.lI/t "now '10 AN‘I‘II1l()TI(‘ Rl:‘.S‘I.S"I'AN(‘l;‘
`
`33/
`
`donuclcast: digestion and substituting the neo
`fragment via the corresponding cohesive ends.
`The pSV3—nco and pSV5-nco (Fig. 2) deriva-
`tives were constructed from pSV2-neo as pre-
`viously described (8.25). Each of the pSV-neo
`plasmids replicates efficiently in E.
`("U/f 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 UK’ 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-
`lated 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), pSV2-3G
`(25) DNA, or mock-transfected without DNA
`have never yielded G418-resistant colonies
`(frequency less than 1
`transformant
`in 10’
`transfected cells).
`In contrast to the low frequencies of G418-
`resistant transformation with pRZll2 (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
`
`plasmids 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 G4l8-
`resistant mouse cell transformants. This differ-
`
`J. Mull. /l[|[lf. G:'Itr'I.. V0]. I. No. 4.
`
`IUN3
`
`HIMI
`
`Hmrllll
`SW”
`
`Still
`
`H/mllll
`Bqlll
`
`—
`Hnul
`
`5
`iAir3
`I
`I
`l<— neo —a—l
`
`pRZ'l02
`
`pRZll2
`
`IHHlCllJ Him/III
`
`Hmcll
`'81]/II
`I
`i<— neo —>i
`“G
`5
`
`i(
`
`- g
`
`Hp.Il
`
`lHim.'lI CLEAVAGE
`
`Hmrll II
`
`
`
`
`
`I89/llHmcll Hincllat
`
`EamHl LINKERS
`B.miHl + H/ntllll CLEAVAGE
`ISOLATION OF neo GENE FHAGMENT
`
`Hrndl“
`Eqlll
`
`b‘.:mH|
`
`INSERTION mro pen322
`SELECTION FOR Amp",~eo"
`
`Hindi I I
`By! I I
`
`Ecaneo
`
`’IG. 1. Organization of transposon Tn5 and scheme tor the
`onstruction of recombinant plasmid pBFl-neo. The plasmid
`uFlZ102 contains a complete copy ol Tns inserted into Co|E1.
`‘he inverted repeat sequences at the ends of the transposon
`re shown as thick lines. The region essential tor expression
`If neomycin resistance in E. coli (neo) is indicated together
`till) the ATG codon that initiates the coding sequence of the
`ihosphotranslerase. Relevant restriction endonuclease rec-
`lgl'l|llOn sites are included in the diagram. pRZ112 was de-
`Wed from pFtZ102 by partial digestion with Hincll endonu-
`lease and ligation to eliminate a large segment of Tn5 DNA to
`ne 3‘ side of neo (Hincll endonuclease cleaves at Salt and
`‘pal endonuclease recognition sites.) Details of the ma-
`ipulatlons involved with the construction of pBFl-neo are
`liven in the text.
`
`Ieomycin: 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
`rum pSV2-BC (25) with Hindlll and Bglll en-
`
`Sanofi/Regeneron Ex. 1004, pg 68
`
`

`
`.
`
`.S'()UTHI'fRN A.'\’I) I’. BI-JRG
`
`pBR32Z on
`
`pSV2- neo
`
`Pill’
`
`B.lrnHi
`
`Jr;-mil ll
`
`pBFl322 on
`
`s=BF|-‘£22 on
`
`FIG. 2. Structures of the QSV-neo hybrid plasmids. The oSV plasmids are composed of DNA segments from various source
`pBH322 DNA, represented by the solid black are. contains the p8Fl!-I22 origin 01 DNA replication (pBR322 on) and the /3-Iactarna
`gene (Arrlp"l: the hatched segment represents the neo gene H.-1 Kb fragment. Fig. 1); SV40 DNA sequences are indicated as It
`dotted segments. The SWO origin 0! DNA replication (SWO on) and the sv4o early promoter are present on a small fragma
`{SWO map units {).?'1 41.65] immediately 5' to the new segment. The plasmids pSV3-neo and pSV5-neo were constructed lro
`pSV2-neo by insertion ol either an intact SV-to early region or an intact polyoma early region that contains a duplication or H
`viral origin of replication [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
`transformations with pSV3-g'pt and pSV5-gpt
`recombinants (26).
`
`Stable Integration of neo DNA Sequence:
`after Transfection into Cultured Cells
`
`The content and organization of neo DN}
`sequences have been examined in a number 0
`stable G418 transformed cell lines. The Ltk' 0
`
`TABLE 2. Tru/r.\_/iirmulin/i _/i'cqiu'm't'('.\' flu‘ I'¢'t'uIIlbimi/u
`Hon pIu.\'/iiid.\' "
`
`Host cells
`
`Ltk
`
`3'l‘(i
`
`TC7
`
`I x In '
`7 x I0
`~3 X I0 ‘
`pSV2~ncu
`6 x It)
`-"
`|.5 x I0 ’
`--3 X Ill
`'
`[ISVJ-ncu
`2 x in '
`4 x It)
`-
`-3 x It)
`'
`pSV5-nco
`Nlll.‘UlLlI1lL‘.HlMI[il|Cd
`I X It)
`5 X It) "
`pBR-nut»
`"
` Z_
`
`" 'l‘ran.~;l'urmalitin lrcqucncy is expressed as the fraction ulculls plated
`that product: viahlc colonic.» in §L‘lL‘CllVl.' medium llillowing tramilbctiun
`with saturating levels of IJNA.
`
`J. Mu]. Am)/. (ii-m'I..
`
`l'uL I, No. 4, I982
`
`Sanofi/Regeneron Ex. 1004, pg 69
`
`

`
`TRA NSF()RMATI()N TO /\NTlBI()'I'I(‘ RE.S'IS'l'AN('l;'
`
`(To transformants arising from transfcctions
`;.:[ih pSV2—neo. pSV3—nco. or pSV5—neo do not
`contain plasmid—rclated sequences (limits of
`detection 0.5 plasmid molecules per cell) in the
`low molecular weight DNA of a Hirt superna-
`tant (47). However. cell DNA obtained from
`G418-resistant transformants contained nucle-
`“ride sequences homologous to the transfecting
`plasmid DNA. High molecular weight DNA
`from a representative set of pSV2—neo Ltk'
`transformants was cleaved with Er-nRl 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 E('0Rl restriction site, each
`integrated plasmid copy should produce two
`hands after hybridization with the pSV2—neo
`probe if there is no rearrangement or scram-
`bling of the plasmid DNA sequences during in-
`iegration. 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-
`nco DNA is produced by digestion with Ec'0Rl
`cndonuclease (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-
`taining G418 (200 pig/ml): such cells remain
`fully resistant
`to G418 when challenged in
`
`Ltk ‘lpSV2neo
`
`—
`
`UNEAR
`— pSV2neo
`
`FIG. 3. Detection of pSV2—neo DNA sequences in trans-
`lormed cells. High molecular weight cell DNA was extracted
`from representative G413-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 than fractionated by electrophoresis in a
`0.6% agarosa 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 tor
`Eco RI 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 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. Mu]. r|[‘[’i. G(‘Il(‘I.. Vol. I. Nil, 4. I93.’
`
`Sanofi/Regeneron Ex. 1004, pg 70
`
`

`
`I’. J. SOUTHERN AND P. BERG’
`
`Ltk ‘ pSV2neo2F
`
`Ltk" pSV2nao2G
`
`Ltk‘pSV2neo2G
`
`FIG. 4. Detection of pSV2-neo DNA se-
`quences alter extended passaging of
`transformed cells. High molecular weight
`chromosomal DNA was extracted from
`parallel cultures of the translormed cell
`lines 2F and 2G that had been maintained
`in the presence (+G416) 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 samples 0i
`the original DNA preparations (see Fig.3).
`The central track contains parental Ltk‘
`cell DNA. The panel at the right ol the tig-
`ure is a shorter exposure during au-
`toradiography ol the Ltk‘ pSV2-neo 2G
`cell DNA.
`
`ble during growth ofthcse 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-
`conflucnt 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
`IO“ to l0"‘ whereas the second set yielded
`about one-fifth the number of transformants
`predicted from the initial value and the number
`ofcell 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. Mnl. A/I/II. (ir'n¢'I.. Vol. I. No. 4. I‘M2
`
`seems that the neo marker is associated will
`the cellular genome relatively early and is re
`taincd 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 phosphotmnsfcrasc
`protein. Cytoplasmic. poly(A)* 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 ugurose gel (39.40) yielded it singlfi
`fragment of I500 bases (Fig. 5). The production
`of a I500 base fragment is consistent with the
`occurrencc ofan RNA that extends throughout
`the length ofthc neo segment and suggests thal
`
`Sanofi/Regeneron Ex. 1004, pg 71
`
`

`
`IR/t.\’.$'I"()R.'lI.-tHUN H) AN’!/I!l()'Il('RESlS7}1NCli
`
`MARKERS{K
`
`FIG. 5. Detection oi neo sequences in cyto-
`plasmic RNA extracted from pSV2-neo trans-
`formed Ltk‘ cells. A DNA probe was prepared by
`5‘ end-labeling with "P at the Berni-tl restriction
`site at the 3’ extremity oi the neo DNA fragment.
`(The BamHI site was not preserved in the con-
`struction of pSV2-neo and the probe lor this ex-
`periment was prepared from 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
`egarose get. Marker bands were produced by
`digesting the DNA probe with Bgll. Hindlll and
`Bgilt restriction endonucleases. The DNA frag-
`ment protected by the transformed cell FtNAs is
`shown in the diagram as a continuous line
`under the representation oi the DNA probe.
`
`PROBE FOR pSV2-neo RNA
`
`Hull Him/Ill
`: :
`72
`
`320
`
`Fvull
`290
`
`Halli
`
`EcoFll
`
`oessnvso rnorecrso
`FHAGMENT
`
`1500
`
`the 5'-end of the RNA is located beyond the
`Hmdlll site. most probably in proximity to the
`S\'4ll 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-
`lurincd cell lines is about the same.
`lo determine if the Cr-l|8-resistant transfor-
`
`m..nts produce phosphotransfcrase protein,
`-cnticonflucnt cultures of normal and trans-
`tnrmcd cells were labeled for l4 h at 37°C with
`llllcucinc. Labeled extracts of E. ('0/i har-
`Nnng pBR-nco or pBR322 were made for
`mmpurison (see ltrlateriatls and Methods). Im-
`munc complexes prepared from each of these
`ntracrs were clcctrophorcscd in sodium dode-
`cyl sulphate polyarcrylamidc gels (Fig. 6). it is
`evident that the immunoprccipitatc from mli
`‘f")’ing pBR-neo contains ti peptide of 25
`Ktlodultuns. WhCi'CdS the extract from cells
`“Kilns neo does not contain that peptide. The
`
`molecular weight estimate for APH(3’)lI ob-
`tained from E. coll 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 ofa 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 20 than 2F.
`the
`amount of phosphotransfcrase peptide appears
`to be about the same. even somewhat greater in
`EF‘.
`the same disparity occurs between neo
`DNA copy number and neo mRNA (see Fig.
`5). This suggests that many ofthe neo genes in
`isolate 2G may not be expressed.
`
`.1, Mill. .-l/Ipl. Gr“Ilt'I.. Vol. I.
`
`.-\/u.
`
`-1.
`
`I9-"3
`
`Sanofi/Regeneron