%*“>URNAL OF
`WOLECULAR
`AND APPLIED
`“ENETICS
`
`
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`/OLUIVIF P1-|\|Ul\/IBEP Z1-198%?-PAVEN PPESS
`
`Genzyme Ex. 1004, pg 62
`
`

`
`Journal of
`Molecular and Applied Genetics
`
`Editor-in-Chief:
`
`Howard M. Goodman, Ph.D.
`
`Depamnent of Molecular Biology
`Massachusetts General Hospital
`Boston, Massachusetts 02114, U.S.A.
`
`RECEIVED
`
`J01. 14 1982
`
`263
`
`273
`
`289
`
`301
`
`315
`
`Volume 1, Number 4, 1982 Blomedlul Llbflry ucsp I
`
`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
`
`327
`
`Transformation of Mammalian Cells to Antibiotic
`Resistance with 8 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.
`
`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 City
`Gary Gussin, Iowa City
`Dean H. Hamer, Bethesda
`Masayori Inouye, Stony Brook
`Robert Kamen, London
`Yuet Wai Kan, Sun FI’(tll('l.\‘(‘()
`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. Steitz, New Haven
`Robert T. Tjian, Berkeley
`Michael Wigler, Cold Spring Harbor
`Patricia Zambryski. Sari 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
`lrene Rublnson, Advertising Manager.
`Raven Press. New York
`
`to the
`
`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 mtesfor Vol. I, 198ll2: 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 63
`
`Genzyme Ex. 1004, pg 63
`
`

`
`Jourlllll 0ftWUl1't'II/tlr (UNI A/J/I/lt'd Ut'IIl‘I1(.\
`1:327-J-3|
`'t'v I983 Raven Press, 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
`
`Dt'[)(lI‘fI)1('l1I ofBim'lt¢'Ini.vII'v. Stanford UI1i|'(‘1‘.\'ll_\' Mc'di('u/ C(‘I1l(’I'. Stmtfnrrl. C'u/ffornia. 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.
`In 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—Cel1 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
`(3-10). Alternatively. exogenous DNA may be
`introduced directly into recipient cells by either
`the calcium phosphate precipitation technique
`
`Reccivedjanuary 4. 1982: accepted February 18. 1982.
`Address correspondence and reprint requests to Dr.
`7.1. Southern. Department of lmmunopathology.
`gsfripps Clinic and Research Foundation, La Julla. CA
`.037.
`
`327
`
`(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 64
`
`Genzyme Ex. 1004, pg 64
`
`

`
`338
`
`P. .I.
`
`.S'()(/'l‘III-IRN AN!) P. BERG
`
`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-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, G4l8 (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. Mal. A/ml. (p':'Iu'I..
`
`l/ul. I. No. 4. I982
`
`Brenner) that the acquisition and expression 0
`the phosphotransferase gene by mammaliar
`cells might ctunfcrfesistzince to G4l8 toxicity
`Accordingly.
`the phosphotransferase gang
`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 SV4t) early promoter is 5'-proximal.
`and an intron and polyadenylation signal are
`3'-proximal.
`to the neo gene (8.25). Transfeo
`tion of a wide variety of mammalian ceil lines
`with these pSV-neo recombinants yields stable
`transformants that are resistant to G-H8 at a
`
`frequency of one transformant per l0‘-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
`0418 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 G4f8 (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 I0"/c newborn calf serum.
`penicillin and streptomycin. and the indicated
`concentrations of G418 were added to [ht
`medium.
`
`Enzymes
`
`Restriction enzymes were purchased from
`New England Biolabs and Bethesda Research
`Laboratories and digestions were performed
`according to the supplier's specifications. T‘
`polynucleotide kinase was purchased l'I'0"‘
`New England Nuclear and SI nuclease front
`Boehringer Mannheim. T4 DNA ligase and 5-
`mli DNA polymerase l were kindly provided
`by S. Schcrcr. Stanford University.
`
`Genzyme Ex. 1004, pg 65
`
`Genzyme Ex. 1004, pg 65
`
`

`
`'I'RAN.S'l-'()R.\M 'll()N 'I()x1N7'IBl()'I'l(' Rl;'SI.S"l'ANC'l-.‘
`
`329
`
`Antibiotic G418
`
`Samples of antibiotic G418 were generously
`provided by Dr. P. J. L. Daniels of Schering
`Corporation. Stock solutions containing 4
`mg/ml G418 in 100 mM N-2-hydroxyethylpi-
`pcrazine-N’-2’-ethanesulphonic acid buffer. pH
`7_3, were stored at —20°C and added in ap-
`propriilte 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 p.g for approximately 5 X 10"
`:ells) 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
`
`100 agmt. 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-
`electively 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
`;on1c instances,
`the initial selection and sub-
`
`tloning used 400 ug/ml of G418 but the trans-
`ormed cells were subsequently maintained in
`‘.00 ug/ml of G418.
`The selection strategy of permitting cell
`trowth prior to the addition of G418 was
`idopted because a significant reduction in the
`ransformation frequency occurred if G418 was
`aided before 48 h. The trapsfected 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. (17),
`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 [y-"2P]-
`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/. App]. (a'um‘r., Vol. I. No. 4. 1933
`
`Genzyme Ex. 1004, pg 66
`
`Genzyme Ex. 1004, pg 66
`
`

`
`_?_i’()
`
`P. J. .S'()UTIIl:'RN AN!) I’. Bl-{RC
`
`with ["'H]leucine (200 ,uCi/plate. specific activ-
`ity 55 Ci/mmol. New England Nuclear Labo-
`ratory). The soluble proteins were extracted
`from approximately 2 X 107 cells (7) and im-
`munoprecipitated with an antiphosphotransfer-
`ase APH(3’)ll serum (provided by J. Davies.
`Geneva). Esc/icricliia (‘vii cells (HBl0l) 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 Ieucine and then [“H]leu-
`cine was added (200 ;LCi/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 14,000g 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. aureus 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“HANCE (New England Nu-
`clear Laboratory). dried and autoradiographed
`as described above.
`
`RESULTS
`
`Mammalian Cells Are Sensitive to 0418
`
`The sensitivity of various cultured cell lines
`to G4l8 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 p.g/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 (100
`;ugJm|) there is a significant delay but the cells
`are killed eventually. All of the cell lines that
`
`J. Mal. Appl. (irm*!.. Vol. I, Nil. 4. I982
`
`TABLE I.
`
`(I4I8-.\'i-/I.\’iIii'i- niunmmliuii rvll li'mx"
`
`Monkey
`
`(‘V1
`CV I -P
`'l‘C7
`COS
`
`Human
`
`LNSV
`He La
`K-56'.’
`
`Mouse
`
`L
`Ltk
`3T3
`3T6
`PCC4
`F9
`MEL
`
`"So far. no cell
`resistant to G-H8.
`
`line has been found that is naturally
`
`have been tested (Table l) are killed by G4l8
`but CVI and HeLa are unusual because. at
`
`high cell density, these cells may require 10- I4
`days in G418 (400 ,ug/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 segment
`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 dccanucleotide se-
`quence containing the BumHl restriction silt-’
`(Collaborative Research) to the ends ofthe n60
`segment (46) the mixture was digested with an
`excess of Bu/nHl and Hindi]! restriction cf)‘
`donucleases and the resulting l.4 kb neo DNA
`fragment was purified by gel electrophoresis-
`This fragment. containing Ilindlll and BumH|
`cohesive ends at the 5’- and 3'-ends, re$P¢“
`tivcly. was inserted between the Hindlll and
`ziiimat restriction sites in p13R322 DNA. Ths
`resulting plasmid, pBR-neo (Fig. I). confers re-
`sistance in IS. vuli‘
`to both ainpicillin and
`
`Genzyme Ex. 1004, pg 67
`
`Genzyme Ex. 1004, pg 67
`
`

`
`7RAN.S'I"0RMz\ "HON 'l'() AN'I'I[1'l()Tl(‘ Rl;‘SI.S"I'AN(‘I:'
`
`33/
`
`donuclcasc digestion and substituting the neo
`fragment via the corresponding cohesive ends.
`The pSV3-neo 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. 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 analyses (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-
`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-,BG
`(25) DNA, or mock-transfected without DNA
`
`have never yielded G418-resistant colonies
`(frequency less than I
`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-
`quency of stable G418-resistant monkey cell
`transformants is two- to three-fold lower with
`
`pSV3-neo than with pSV5-neo. whereas the
`converse applies to the formation of G418-
`resistant mouse cell transformants. This differ-
`
`.I. Mal. /\[l[lf. (h'm'I.. Vol. I. No. 4. I933
`
`Genzyme Ex. 1004, pg 68
`
`Hrml
`; i _
`
`HI/l(/iii
`Sail
`EH/ll
`1
`iAit3
`'
`‘
`l<— neo —o|
`
`
`Hmrl|—||
`Bglll
`I
`pRZ'l02
`
`H;J.i|
`‘H I
`
`/HHICIU Hm;/III
`Hpul
`lat)/II
`
`- ‘
`
`Hincll
`I
`
`i«
`
`M6
`5
`i<- neo —>t
`
`pRZ112
`
`lHiIlt.‘i| CLEAVAGE
`
`Hindi I I
`
`
`
`Hincll HincllBaal'89/ll
`
`
`
`HanlHl LINKEHS
`BJIHHI + Hinrllll CLEAVAGE
`ISOLATION OF neo GENE FRAGMENT
`
`Him‘/III
`‘Salli
`
`B.rmH|
`
`wsermow mro pen322
`SELECTION FOFI AmpR,NeoR
`
`F“
`
`Hinrllll
`Bglll
`
`
`
`iIG. 1. Organization of transposon Tn5 ‘and scheme for the
`onstruction of recombinant plasmid pBFl-neo. The plasmid
`tFlZ102 contains a complete copy of Tn5 inserted into CoIE1.
`‘he inverted repeat sequences at the ends of the transposon
`;re shown as thick lines. The region essential for expression
`Ii neomycin resistance in E. coli (neo) is indicated together
`aim the ATG codon that initiates the coding sequence of the
`tnosphotransterase. Relevant restriction endonuclease rec-
`Igf'l|liOn sites are included in the diagram. pRZ112 was de-
`lved 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-
`ipulations 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-
`Il0tlC.
`-
`
`The cloned neo DNA segment was readily
`ntroduced into the plasmid vector—pSV2 (8)
`Fig. 2) by excising the B-globin cDNA segment
`rom pSV’_’-BC (25) with Hindlll and Bglll en-
`
`Genzyme Ex. 1004, pg 68
`
`

`
`333
`
`1’.
`
`.1. sot/7‘:/I-.‘R.-\-' AN!) I’. BI-JRG
`
`Amp
`
`(185132? on
`
`Pvull
`
`pSV2- neo
`
`Pill "
`
`8.miHl
`
`SV40 on
`UN
`
`panazz on
`
`pm,“
`
`s\/40 0,.
`
`0.17
`
`pSV3- neo
`
`fl'airiHl
`
`SV40— Tag
`
`989322 on
`
`Pvull
`
`SV4O on
`
`0.17
`
`PY-
`
`FIG. 2. Structures of the pSV-neo hybrid plasmids. The pSV plasmids are composed of DNA segments from various source
`pBH322 DNA. represented by the solid black are. contains the pal’-‘i322 origin of DNA replication (pBR322 ori) and the ti-Iactama
`gene (Arnp"}: the hatched segment represents the neo gene (1.4 Kb fragment. Fig. 1);SV-10 DNA sequences are indicated as II
`dotted segments. The Swap origin ol DNA replication (SWO on) and the SV40 early promoter are present on a small tragme
`{SWO map units 0.71-0.65] immediately 5' to the neo segment. The plasmids pSV3-neo and pSV5-neo were constructed lro
`pSV2-neo by insertion ol either an intact SV-$0 early region or an intact polyoma early region that contains a duplication of It
`viral origin of replication (Bl.
`
`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-gpt and pSV5-gpt
`recombinflnls (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 G4l8 transformed cell lines. The Ltk’ 0
`
`TABLE 2. Truri.\_/imnulimi _/i'('([m'Il('l'(‘.\’ _/in‘ I’('(‘tIIIlbiIl(lIlI
`Il('n pIu.mriil.\"‘
`
`Hiisi cells
`
`Ltk
`
`3'l‘(i
`
`TC7
`
`'
`I x It)
`7 X It)
`‘
`-3 x It)
`pSV2~ncu
`"’
`6 X It)
`'
`|.5 x Ill
`'
`--3 X ll)
`pSV3-neo
`'
`2 x H)
`'
`4 x It)
`'
`-3 X It)
`pSV5-neo
`
`
`
`
`5 X Ill “ I X It) "[JBR-DL'0 Ni) colonies isul:iIci.I
`
`lrainslormailiun lrcqiicncy is cxprcssctl us the fraction ulcclls plrilcd
`"
`that produce: viiihlc colonies in scluclivi: medium Following trtiiislbctiun
`with saiturziting levels of DNA.
`
`J. Mill.
`
`.-\/I/)/. (:'t'm'I.. Vol. I, No. 4, I982
`
`Genzyme Ex. 1004, pg 69
`
`Genzyme Ex. 1004, pg 69
`
`

`
`TRANSFORMA TION T0 /tNTlBI()'I'l(‘ RESlS'l'AN('l;'
`
`333
`
`Ltk‘/pSV2neo
`/
`
`l2F|2Gl2J|2K| MI
`
`_
`
`LINEAR
`_ pSV2neo
`
`,
`
`C&
`
`20 kb -
`
`_
`
`5'6 kb
`
`1 kb ~
`
`FIG. 3. Detection of pSV2-neo DNA sequences in trans-
`formed cells. High molecular weight cell DNA was extracted
`irom 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
`0.6% agarose gel. DNA in the gel was translerred to DBM
`paper and hybridized with radioactively labeled pSV2-neo
`DNA. There is a single recognition site in pSV2-neo DNA tor
`EcoFil cleavage and the marker track (M) shows the position
`of pSV2-neo linear DNA. Approximate molecular sizes were
`derived irom 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
`
`1T6 transformanls arising from transfections
`(vim pSV2-neo. pSV3-neo, or pSV5—neo do not
`contain plasmid-related sequences (limits of
`detection 0.5 plasmid molecules per cell) in the
`[ow molecular weight DNA of a Hirt superna-
`mnt (47). However. cell DNA obtained from
`G418-resistant transformants contained nucle-
`utide 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-oRl 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-
`tegration. 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 EcoRl
`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 20 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 2G cell
`
`lines have been
`
`cultured for over 3 months (approximately 100
`cell generations) in medium lacking or con-
`taining G4l8 (200 ,ugJml): such cells remain
`fully resistant to G418 when challenged in
`
`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. Mul. rl/vpl. C(‘Il(‘I.. Vol. I. Na. 4. I98.’
`
`Genzyme Ex. 1004, pg 70
`
`Genzyme Ex. 1004, pg 70
`
`

`
`334
`
`P. J. SOUTHERN AND P. BERG
`
`Ltk' pSV2neo2F
`
`Ltk" pSV2nao2G
`
`Ltk‘pSV2neo2G
`
`‘:45
`.3:
`-(5
`.3:
`,
`Q,
`Q,
`C’
`0
`0
`0°‘
`G‘
`05
`8.
`5-.‘
`§\
`14‘;/°|G|V|* |’|¢t I‘ I’Iq'I
`
`°
`
`‘
`VIII‘-O‘
`
`FIG. 4. Detection of pSV2-neo DNA se-
`quences after extended passaging oi
`transformed cells. High molecular weight
`chromosomal DNA was extracted from
`parallel cultures of the translormed cell
`lines 2F and 26 that had been maintained
`in the presence (+G-118) or absence
`(—G418) oi G418 (see text). DNA samples
`were digested with EcoFtl endonuclease
`and analyzed as described in Fig. 3. The
`tracks at the extremities ol the gel
`_
`(labeled 2F and 26) contain samples of
`the original DNA preparations (see Fig. 3). R
`The central track contains parental Ltk‘
`cell DNA. The panel at the right of the tig-
`ure is a shorter exposure during au-
`toradiography of the Ltk‘ pSV2-neo 2G
`cell DNA.
`
`"""
`
`-
`
`"’
`
`pSV2ne
`
`_ LINEA
`
`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
`l0‘* to l0""' 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 G4l8 and considering the
`inherent
`inaccuracies of the experiment,
`it
`
`.1. Mnl. A/I/II. (im¢'I., Vol. I. No. 4. I932
`
`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, 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 SC‘
`quenccs.
`In each sample. digestion with SI
`nuclease and electrophoresis of the protected
`DNA in an ngarose gel (39.40) yielded ll single
`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 that
`
`Genzyme Ex. 1004, pg 71
`
`Genzyme Ex. 1004, pg 71
`
`

`
`/It/t.\'.s'I-‘ii/e.i1.4 "I/UN 'l() AN’!/11/()1/(' Rl;'SIS'I"AN(‘l;‘
`
`335
`
`MARKERS
`
`Ltk‘
`
`2F
`
`2G
`
`Bg/ll
`
`— PROBE
`
`bp
`
`FIG. 5. Detection ol 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 BamHI restriction
`site at the 3’ extremity oi the neo DNA tragment.
`(The BamHI site was not preserved in the con-
`struction ol pSV2-neo and the probe tor 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
`Bgilt restriction endonucleases. The DNA trag-
`ment protected by the translormed cell FtNAs is
`shown in the diagram as a continuous line
`under the representation ot the DNA probe.
`
`PROBE FOR pSV2-neo RNA
`
`EcoR|
`
`Pirull
`290
`
`Egil Hindlll
`:
`:
`
`"I2
`
`320
`
`Sqfll
`
`1110
`
`BamHl
`
`osssnvso PROTECTED
`FRAGMENT
`
`
`1500
`
`the 5'-end of the RNA is located beyond the
`Hmdlll site. most probably in proximity to the
`S\'-til 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-
`lorincd cell lines is about the same.
`
`"In determine if the G-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
`
`llllcucine. Labeled extracts of E. coli har-
`
`Nnng pBR-neo or pBR322 were made for
`mmpurison (see Mztterizils and Methods). Im-
`mune complexes prepared from each of these
`cttnacts were elcctrophoresed in sodium dode-
`cyl sulphate polyacrylamidc gels (Fig. 6). ll is
`evident that the immunoprecipitate from r-nli
`‘fffying pBR-neo contains it peptide of 25
`Ktlodultons. whereas the extract from cells
`hiking neo does not contain that peptide. The
`
`molecular weight estimate for API-l(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