United States Patent ll9J
`Axel et al.
`
`[54] PROCESSES FOR INSERTING DNA INTO
`EUCARYOTIC CELLS AND FOR
`PRODUCING PROTEINACEOUS
`MATERIALS
`
`[75]
`
`Inventors: Richard Axel, New York; Michael H.
`Wigler, Cold Spring Harbor; Saul J.
`Silverstein, Irvington, all of N.Y.
`
`[73] Assignee: The Trustees of Columbia University,
`New York, N.Y.
`[21] AppL No.: 124,513
`[22] Filed:
`Feb. 25, 1980
`[51]
`Int. CJ,J ....................... C12N 15/00; Cl2N 5/00;
`C12P 21/00; C12Q 1/68; C12Q 1/02; C12Q
`1/04
`[52] u.s. a ......................................... 435/6; 435/172;
`435/240; 435/317; 435/811; 435/948; 435/29;
`435/34; 435/68
`[58] Field of Search ................... 435/68, 172, 70, 240,
`435/241,948, 811, 6, 29, 34; 424/85, 177, 178,
`180
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,800,035
`J/1974 Goore ................................... 435/68
`4,195,125 3/1980 Wacker ............................... 435/172
`
`FOREIGN PATENT DOCUMENTS
`2010847 7/1979 United Kingdom .................. 435171
`
`OTHER PUBLICATIONS
`Kretschmer et aL, J. Bacteriology 124, 225-231 (1975).
`Wigler, Cell 11, 223-232 (1977).
`Mantei et aL, Nature 281, 40-46 (1979).
`Lai et aL, PNAS 77(1), 244-248 (Jan. 1980).
`Wigler et al., Cell 16, 777-785 (1979).
`Wigler et aL, Eucaryotic Gene Regulation Proc. Inc(cid:173)
`.-UCLA Symposia, R. Axel and T. Maniatis, Editors,
`·
`Academic Press, 457-475 (1979).
`Wigler et al., PNAS 76(3), 1373-1376 (1979).
`Wold eta!.. PNAS 76(11), 5684--5688 (1979).
`The Merck Index, 8th Edition, Merck & Co., Inc., Rah(cid:173)
`way, N.J., 568 (1968).
`Szybalska et al., PNAS 48, 2026-2034 (1962).
`
`[II]
`
`[45]
`
`4,399,216
`Aug. 16, 1983
`
`McCutchan et aL, Journal National Cancer Institute, 41,
`351-356 (1968).
`Wigler et aL, Cell 14, 725-731 (1978).
`Willecke et aL, Malec. Gen. Genet., 170, 179-185
`(1979).
`Graf et aL, Somatic Cell Genetics 5, 1031-1044 (1979).
`Pellicer et aL, Ce1114, 131-141 (1978).
`Mercola et aL, Science 208, 1033-1035 (May 1980).
`Wahl et aL, J. Bioi. Chern., 254(17), 8679-8689 (Sep. 10,
`1979).
`Anderson et al., Scientific American, 245(1), Jul. 1981,
`106-121.
`Fox. C & E News, 35, 36, 42, Sep. 29, 1980.
`Marx, Science, 208, 386-387, Apr. 1980.
`Begley, Newsweek, 80. Apr. 21, 1980.
`Miller et aL, PNAS 75(7), 3346-3350 (1978).
`Primary Examiner-l:<:sther M. Kepplinger
`Attorney, Agent, or Firm-John P. White
`[57]
`ABSTRACT
`The present invention relates to processes for inserting
`DNA into eucaryotic cells, particularly DNA which
`includes a gene or genes coding for desired protein(cid:173)
`aceous materials for which no selective criteria exist.
`The insertion of such DNA molecules is accomplished
`by cotransforming eucaryotic cells with such DNA
`together with a second DNA which corresponds to a
`gene coding for a selectable marker.
`The invention further relates to processes for inserting
`into eucaryotic cells a multiplicity of DNA molecules
`including genes coding for desired proteinaceous mate(cid:173)
`rials by cotransformation with the desired genes and
`with amplifiable genes for a dominant selectable marker
`in the presence of successively higher amounts of an
`inhibitor. Alternatively, the insertion of multiple copies
`of desired genes is accomplished by transformation
`using DNA molecules formed by ligating a DNA mole(cid:173)
`cule including the desired gene to a DNA molecule
`which includes an amplifiable gene coding for a domi(cid:173)
`nant selectable phenotype such as a gene associated
`with resistance to a drug in the presence of successively
`higher amounts of an agent such as a drug against which
`the gene confers resistance so that only those eucaryotic
`cells into which multiple copies of the amplifiable gene
`have been inserted survive.
`
`73 Claims, 2 Drawing Figures
`
`Sanofi/Regeneron Ex. 1018, pg 549
`
`Merck Ex. 1018, pg 575
`
`

`
`U.S. Patent Aug. 16, 1983
`COTRAN SFORMATION OF EUCARYOTIC CELLS
`
`Sheet 1 of 2
`
`4,399,216
`
`~
`DNA I CODINC:I FOR
`DESIRED PROTEINACEOUS
`MATE"R!AL..
`
`+
`
`DNA II C.CD\NG ~0~
`SEL.E(. TABLE
`MARKEI2
`(tK+)
`
`NUCLEUS
`(Hl(OMOSONAL DNA
`EUCAR.YOTIC C.ELL(tK-)
`
`IN PRESENCE
`OF SELECTIVE CRITERIA (HAT)
`
`j COTRANSFORM
`
`EUCARVOTIC CELL (tK-t)
`____ __.___ DNA rt
`
`DNA I
`
`CULTURe IN PRESENCE' OF
`SE"LECTWE CRITERIA (HAT)
`
`-[§\
`MATERIAL~
`
`DESIROD
`
`m_OESIRED
`~ I(ECOVERY
`MAiERlAL
`
`DESIRED MATERIAL
`(e.~. INTERFERON, INSULIN, ETC.)
`
`Sanofi/Regeneron Ex. 1018, pg 550
`
`Merck Ex. 1018, pg 576
`
`

`
`U.S. Patent Aug. 16, 1983
`
`Sheet 2 of 2
`
`4,399,216
`
`RESCUE OF p5R FROM TRANSFORMED MOUSE CELLS
`
`HIND m CLEAVED pBR 3'2'2.
`
`l COTRANSFORH MOUSE CELLS
`' Xbo. r
`
`• 0
`pBR 322 INTEGRATED \N MOUSE CNA
`
`t
`'Xba. I
`
`j CLEAVE WITH Xba. I
`
`CIRCULARI'ZE WITH UGASE
`Tr<ANSFCI?H E,COLl X 1776
`
`MOUSE DNA !fiJ SERT
`
`'OACTe:RlAL
`SCHEME FOR THE RESCUE OF
`PLAS.Ml DS FROM TRAN5FORMED CU L TlJRED
`CElLS USI~G DOUBLE" SELECTION TEC...HNIQUES,
`
`Sanofi/Regeneron Ex. 1018, pg 551
`
`Merck Ex. 1018, pg 577
`
`

`
`PROCESSES FOR INSERTING DNA INTO
`EUCARYOTIC CELLS AND FOR PRODUCING
`PROTEINACEOUS MATERIALS
`
`The invention described herein was made in the
`course of work under grants numbers CA-23767 and
`CA-76346 from the National Institutes of Health, De(cid:173)
`partment of Health and Human Services.
`
`1
`
`4,399,216
`
`2
`formation that it was not possible to distinguish tranfor(cid:173)
`mants from cells which had merely undergone sponta(cid:173)
`neous reversion. Again, as with procaryotic cells, fur(cid:173)
`ther. reports of eucaryotic transformation occurred in
`the literature, but such results were oftentimes not re(cid:173)
`producible by others. In addition, low frequencies of
`transformation, lack of understanding of the molecular
`basis for gene expression and the lack of molecular
`hybridization probes contributed to the lack of progress
`10 in this area. As a result, studies on the transformation of
`FIELD OF THE INVENTION
`eucaryotic cells were essentially restricted to viral
`genes. Graham, F. L., et al., Cold Spring Harbor Symp.
`This invention concerns the introduction and expres-
`sian of genetic informational material, i.e., DNA which
`Quant. Bioi. 39: 637-650 (1975) and McCutchen, J. H.
`and Pagano, J. S., Journal National Cancer Institute, 41:
`includes genes coding for proteinaceous materials and-
`lor genes regulating or otherwise influencing the pro- 15 351-357 (1968).
`More recently, however, eucaryotic cells, specifi-
`duction thereof, into eucaryotic cells, that is, cells of
`organisms classified under the Superkingdom Eucary-
`cally mammalian cells, were transformed with foreign
`otes including organisms of the Plant and Animal King-
`DNA coding for a selectable phenotype. Wigler, M., et
`dams. Such genetic intervention is commonly referred
`al., Cell II: 223-232 (I 977). This work has been ex-
`to as genetic engineering and in certain aspects involves 20 tended and has resulted
`in
`the present invention
`wherein it has been discovered inter alia that eucaryotic
`the use of recombinant DNA technology. The inven-
`tion disclosed is to be distinguished from the introduc-
`cells can be cotransformed to yield transformants hav-
`tion of DNA into organisms of the Superkingdom
`ing foreign DNA integrated into the chromosomal
`DNA of the eucaryotic cell nucleus. Moreover, it has
`Procaryotes including particularly bacteria. This dis-
`tinction is based in part upon the basic differences be- 25 unexpectedly been discovered that such foreign DNA
`tween eucaryotic and procaryotic cells, the former
`can be expressed by the cotransformants to generate
`being characterized by true nuclei formed by nuclear
`functional proteins. In addition, by contrast with pro-
`envelopes and by meiosis and the latter being character-
`caryotic transformants, the foreign DNA is stably ex-
`ized by the absence of well-defined nuclei and the ab-
`pressed through hundreds of generations, a result that
`sence of meiosis. Moreover, at the genetic level many 30 may be attributable to integration of the foreign DNA
`genes in eucaryotes are split by non-coding sequences
`into the chromosomal DNA.
`which are not continuously colinear, whereas in proca-
`The present invention provides major advances over
`ryotes, the genes are continuously colinear.
`bacterial systems for future use in the commercial prep(cid:173)
`aration of proteinaceous materials particlarly proteins
`35 of eucaryotic origin such as interferon protein, antibod(cid:173)
`ies, insulin, and the like. Such advantages include the
`ability to use unaltered genes ceding for precursors for
`such proteinaceous materials. After cellular synthesis,
`the precursor can be further processed or converted
`within the eucaryotic cell to produce the desired mole(cid:173)
`cules of biological significance. This phenomenon is
`well known for insulin which is initially produced in the
`eucaryotic cell as preproinsulin which is then converted
`to active insulin within the cell by appropriate peptide
`cleavage. Since procaryotic cells lack the requisite cel(cid:173)
`lular machinery for converting preproinsulin to insulin,
`the insertion into a procaryotic cell of the eucaryotic
`gene associated with insulin will result in the produc(cid:173)
`tion of preproinsulin, not insulin. Although, in the case
`of insulin, a relatively small and well characterized
`protein, this difficulty can be overcome by chemical
`synthesis of the appropriate gene, such an approach is
`inherently limited by the level of understanding of the
`amino acid sequence of the desired protein. Thus, for
`interferon protein, clotting factors, antibodies and un(cid:173)
`characterized enzymes, for which the exact amino acid
`sequence is not yet known, a procaryotic system will
`likely not prove satisfactory. By contrast, a eucaryotic
`system is not associated with such disadvantages since
`the eucaryotic cell possesses the necessary processing
`machinery. It is thus one important object of the present
`invention to provide a process for producing desired
`proteinaceous materials such as interferon protein, insu(cid:173)
`lin, antibodies and the like which does not require a
`detailed molecular understanding of amino acid se(cid:173)
`quence.
`In addition to the problem of precursors having addi(cid:173)
`tional amino acids which must be removed to produce
`
`BACKGROUND OF THE INVENTION
`Although advances in the understanding of procary(cid:173)
`otic organisms, particularly bacteria, having for the
`most part proceeded independently of advances in the
`understanding of eucaryotic organisms, it may be help-
`ful to an appreciation of the present invention to set 40
`forth certain developments involving procaryotes.
`In 1944, A very reported the transformation of a pro(cid:173)
`caryotic cell using DNA-mediated transfer of a cellular
`gene. Avery, 0. T., et al., J. Exp. Med. 79: 137-158
`(1944). Thereafter, reports of procaryotic transforma- 45
`tion occurred in the literature. In 1975, Cohen and oth-
`ers reported results involving first transformation, then
`cotransformation of the procaryote Escherichia coli.
`Kretschmer, P. J., et al., J. Bacteriology 124: 225-231
`(1975). In the experiments reported therein the authors 50
`disclosed the cotransformation of procaryotic cells
`using plasmid DNA, that is, extrachromosomal DNA
`which occurs naturally in many strains of Enterobac(cid:173)
`teriacae. In these experiments it was found that particu-
`lar cells in a CaCh-treated bacterial population are 55
`preferentially competent for tranformation. However,
`the frequency of transformation and the stability of the
`transformants obtained was low, possibly because the
`plasmid is not incorporated into the chromosomal
`DNA. As a result, cotranformants lost acquired traits 60
`after several generations. In addition, these experiments
`with bacteria: required the addition of a gene promoter
`to the transforming DNA in order to obtain expression.
`Meanwhile, experiments with eucaryotic cells pro(cid:173)
`ceeded substantially independently··.ofthose with pro- 65
`caryotic cells. In 1962, Szybalska, E. H. and Szybalski,
`W. PNAS 48: 2026 (1962) reported the transformation
`ofmammlian cells but with such low frequency of trans-
`
`Sanofi/Regeneron Ex. 1018, pg 552
`
`Merck Ex. 1018, pg 578
`
`

`
`4,399,216
`
`4
`BRIEF DESCRIPTION OF l;HE DRAWINGS
`FIG. 1 is a schematic· flow diagram illustrating the
`cotransformation process in accordance with the pres(cid:173)
`ent invention.
`FIG. 2 is a schematic flow diagram illustrating a
`process for recovering foreign DNA I ·from cotrans(cid:173)
`formed cultured cells using double selection techniques.
`
`3
`active protein, important biological materials may be
`modified by chemical additions after synthesis and
`cleavage. Thus, for ex'!-mple, human-produced inter(cid:173)
`feron is a glycoprotein containing sugar molecules in
`addition to protein. If produced in a bacterial cell, the
`interferon lacks· the sugar molecules which are added
`when interferon is produced in a human cell. Moreover,
`proteinaceous materials produced wiihin bacteria may
`include endotoxins which can cause inflammation if the
`proteinaceous material is administered to a mammal 10
`without significant purification. By contrast, interferon
`produced in a eucaryotic cell would be free of endotox-
`.
`ins.
`It is therefore another important object of this inven(cid:173)
`tion to provide a process for producing compounds 15
`which include both non-proteinaceous. and protein(cid:173)
`aceous moieties such as glycoproteins which cannot be
`produced in bacterial cell.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Prior to setting forth the invention, it may be helpful
`to an understanding thereof to set forth definitions of
`certain terms to be used hereinafter.
`Transformation means the process for changing the
`genotype of a recipient cell mediated by the introduc(cid:173)
`tion of purified DNA. Transformation is typically de(cid:173)
`tected by a stable and heritable change in the phenotype
`<if the recipient cell that results from an alteration in
`20 either the biochemical or morphological properties of
`SUMMARY OF THE INVENTION
`the recipent cell.
`This invention provides a process for inserting for-
`eign DNA into eucaryotic cells by cotransforming the
`Cotransformation means the process for carrying out
`transformations of a recipient cell with more than one
`cells with this foreign DNA and with unlinked DNA
`which codes for proteinaceous material associated with 25 different gene. Cotransformation includes both simulta-
`a selectable phenotype not otherwise expressed by the
`neous and sequential changes in the genotype of a recip-
`cell. The cotransformation is carried out in a suitable
`ient cell mediated by the introduction of DNA corre-
`medium and in the presence of selective conditions
`sponding to either unlinked or linked genes.
`permitting survival and/or identification of eucaryotic
`Proteinaceous material means any biopolymer formed
`cells which have acquired the selectable phenotype. 30 from amino acids.
`The process of this invention is particularly suited for
`Genotype means the genetic constitution of an organ-
`the insertion into eucaryotic cells of DNA which codes
`ism as distinguished from its physical appearance.
`for proteinaceous materials which are not associated
`Phenotype means the observable properties of an or-
`with a selectable phenotype such as interferon protein,
`ganism as produced by the genotype in conjunction
`insulin, growth hormones clotting factors, viral anti- 35 with the environment.
`Selectable phenotype is a phenotype which confers
`gens, antibodies and certain enzymes.
`By use of the cotransformation process of the present
`upon an organism ability to exist under conditions
`invention is it possible to produce eucaryotic cells
`which kill off all organisms not possessing the pheno-
`which synthesize desired proteinaceous and other mate-
`type. Examples include drug resistance or the ability to
`· 1
`d
`h" h
`b
`·
`It
`t
`d.
`40 synthesize some molecule necessary to cell metabolism
`na s an w 1c can e grown m cu ure o pro uce
`in a given growth medium. As used herein, selectable
`these materials in quantities not obtainable with conven-
`phenotypes also include identifiable phenotypes such as
`tiona! technology.
`the production of materials which pass from or are
`In one embodiment of theinvention, the cotransfor-
`secreted by the cell and can be detected as new pheno-
`mation process can be used to insert mtiltiple copies of
`genes coding for desired materials into eucaryotic cells. 45 types either by functional, immunologic or biochemical
`assays.
`Alternatively, a multiplicity of foreign DNA molecules
`. Interferon protein means the proteinaceous part of the
`corresponding to multlp.le copies of a desired gene can
`glycoprotein interferon, that is, the portion remaining
`after removal of the sugar portion. It includes the pro-
`be inserted into eucaryotic cells by transformation with
`molecules each of which is formed by linking a foreign
`tein portion of interferon derived from human Ieuko-
`cyte, fibroblast or Jymphoblastoid cells.
`DNA molecule to a second DNA molecule correspond-
`Chromosomal DNA means the DNA normally associ-
`ing to an amplifiable gene for a dominant selectable
`phenotype not otherwise expressed by the cell. The
`ated with histone in the form of chromosomes residing
`transformation is then carried out in the presence of
`in the nucleus of a eucaryotic cell.
`successively elevated concentrations of an agent per- 55
`Transcription means the formation of a RNA chain in
`mitting survival and/or identification of eucaryotic
`accordance with the genetic information contained in
`the DNA .
`cells which have acquired multiple copies of the am-
`Translation means the process whereby the genetic
`. plifiable gene. This approach is particularly useful when
`information in an mRNA molecule directs the order of
`the dominant selectable phenotype is resistance to a
`drug which is lethal unless multiple copies of the drug 60· specific amino acids during protein synthesis.
`resistant gene are present and the agent is the drug.
`In accordance with. the present invention, foreign
`By inserting multiple copies of genes coding for de-
`DNA I can be inserted into any eucaryotic cell by co-
`sired materials into eucaryotic cells according to either
`transforming the cell with DNA I and with unlinked
`of these approaches it is possible to produce eucaryotic
`foreign DNA II which includes a gene coding for a
`cells which yield desired materials in· high concentra- 65 selectable phenotype not expressed by the cell unless
`acquired by transformation. The cotransformation is
`tions and which can be grown in culture to produce
`carried out in a suitable growth medium and in the
`such materials in quantities not obtainable with conven-
`presence of selective conditions such that the only cells
`·
`·
`tiona! technology.
`
`50
`
`Sanofi/Regeneron Ex. 1018, pg 553
`
`Merck Ex. 1018, pg 579
`
`

`
`4,399,216
`
`5
`which survive or are otherwise altered are those which
`have required the selecta.ble phenotype. See FIG. 1.
`Although the experiments discussed hereinafter con(cid:173)
`cern cultured eucaryotic cells of mammalian origin
`such as human blood cells, mouse fibroblast cells, chin(cid:173)
`ese hamster ovary .cells and mouse teratocarcinoma
`cells, it is clear that the process described is generally
`applicable to all eucaryotic cells including, for example,
`cells from birds such as chickens, cells from yeast and
`fungi, and cells from plants including grains and flow- 10
`ers. Therefore, it is to be understood that the invention
`encompasses all eucaryotic cells even though the inven(cid:173)
`tion may ultimately be most useful in cotransforming
`mammalian cells.
`The present invention is especially useful in connec- 15
`tion with the insertion into eucaryotic cells of foreign
`DNA which includes genes which code for protein(cid:173)
`aceous materials not associated with selectable pheno(cid:173)
`types. Since such proteinaceous materials are character(cid:173)
`ized by the fact that they are not associated with a 20
`selectable phenotype, cells which contain DNA coding
`therefore cannot be identified except by destruction of
`the transformed cell. and examination of its contents.
`Examples of proteinaceus materials, the genes for
`which may be inserted into and expressed by eucaryotic 25
`cells using the cotransformation process include inter(cid:173)
`feron protein, insulin, growth hormones, clotting fac(cid:173)
`tors, viral antigens, enzymes and antibodies.
`Although in some cases the DNA I and DNA II may
`not need to be purified to obtain integration and ex pres- 30
`sion, it is oftentimes preferable that the DNAs be puri(cid:173)
`fied prior to use in cotransforming cells. Such purifica(cid:173)
`tion limits the possibility of spurious results due to the
`presence of contaminants and increases the probability
`that cotransformed cells can be identified and stably 35
`cultured. Also, although not essential, it is sometimes
`desirable that DNA I and/or DNA II have been ob(cid:173)
`tained by restriction endonuclease cleavage of chromo(cid:173)
`somal donor DNAs, such as, for example, restriction
`endonuclease cleavage of eucaryotic chromosomal 40
`DNA. Additionally, it is preferable that DNA I and
`DNA II be treated with calcium phosphate prior to use
`in cotransforming eucaryotic cells. The procedure for
`so treating DNA with calcium phosphate is set forth
`more fully hereinafter. Finally, it is preferable that the 45
`foreign DNA I be present during cotransformation in
`an amount relative to DNA II coding for a selectable
`phenotype which constitutes an excess of the former,
`such as an amount in the range from about I: I to about
`100,000:1.
`In a preferred embodiment of the invention, the for(cid:173)
`eign DNA I and/or the foreign DNA II are attached to
`bacterial plasmid or phage DNA prior to use in cotrans(cid:173)
`forming eucaryotic cells. In a particularly promising
`embodiment, foreign DNA I and/or DNA II are at- 55
`tached to phage DNA and then encapsidated in phage
`particles prior to cotransformation.
`Although any DNA II coding for a selectable pheno(cid:173)
`type would be useful in the cotransformation process of
`the present invention, the experimental details set forth 60
`particularly concern the use of a gene for thymidine
`kinase obtained from herpes simplex virus and the use of
`a gene fon adenine phosphoribosyl transferase. In addi(cid:173)
`tion, a DNA II which includes a gene coding for a
`serectable phenotype associated with drug resistance, 65
`e.g., a mutant dihydrofolate reductase gene which ren(cid:173)
`ders cells resistant to methotrexate greatly extends the
`applicability of the process.
`
`6
`In accordance with a preferred embodiment, the
`cotransformation involves DNA I which is physically
`and chemically unlinked to DNA II, and the DNA I is
`stably integrated into the chromosomal DNA within
`the nucleus of the cotransformed eucaryotic cell.
`Cotransformation in accordance with this invention
`may be carried out in any suitable medium limited only
`in that cotransformed cells be capable of survival and(cid:173)
`/or identification on the medium. Merely by way of
`example, a suitable medium for mouse fibroblast cells
`which have aquired the thymidine kinase gene is HAT
`described more fully hereinafter. Also, the cotransfor(cid:173)
`mation is carried out in the presence of selective condi(cid:173)
`tions which permit survival and/or identification of
`those cells which have acquired the selectable pheno(cid:173)
`type. Such conditions may include the presence of nu-
`trients, drug or other chemical antagonists, temperature
`and the like.
`Eucaryotic cells cotransformed in accordance with
`this invention contain foreign DNA I coding for desired
`materials which can be recovered from the cells using
`techniques well known in the art. Additionally, the cells
`can be permitted to transcribe DNA I to form mRNA
`which in turn is translated to form protein or other
`desired material which may be recovered, again using
`well known techniques. Finally, the cells can be grown
`in culture, harvested and protein or other desired mate(cid:173)
`rial recovered therefrom.
`Although the desired proteinaceous materials identi(cid:173)
`fied hereinabove are natural materials, the process can
`be equally useful in the production of synthetic biopoly(cid:173)
`mers for which synthetic genes are constructed. Thus,
`the instant invention provides a process for producing
`novel proteins not yet in existence. Additionally, it
`provides a process for producing proteins which, al-
`though they presently exist, do so in such minute qunti(cid:173)
`ties or in such impure form that their isolation and/or
`identification cannot otherwise be effected. Finally, the
`invention provides a process for producing partially
`proteinaceous products such as the glycoproteins and
`other products, the synthesis of which is genetically
`directed.
`Another aspect of the invention involves processes
`for inserting multiple copies of genes into eucaryotic
`cells in order to increase the amount of gene product
`formed within the cell. One process for inserting a mul-
`tiplicity of foreign DNA I molecules into a eucaryotic
`cell comprises cotransforming the cell with multiple
`so DNA I molecules and with multiple, unlinked foreign
`DNA II molecules corresponding to multiple copies of
`an amplifiable gene for a dominant selectable phenotype
`not otherwise expressed by the cell. This cotransforma-
`tion process is carried out in a suitable medium and in
`the presence of an agent permitting survival and/or
`identification of cells which acquire the dominant se-
`lectable phenotype. Preferably, this is done in the pres(cid:173)
`ence of successively higher concentrations of such an
`agent so that only those cells acquiring the highest
`number of amplifiable dominant genes (DNA II) sur(cid:173)
`vive and/or are identified. These cells then also contain
`multiple copies of DNA I. This approach is particularly
`appropriate for the insertion of multiple copies of am(cid:173)
`plifiable genes which confer drug resistance upon the
`cell, e.g., the mutant dihydrofolate reductase gene
`which renders cells resistant to methotrexate.
`Cotransformed eucaryotic cells which have acquired
`multiple copies of DNA I may then be used to produce
`
`Sanofi/Regeneron Ex. 1018, pg 554
`
`Merck Ex. 1018, pg 580
`
`

`
`4,399,216
`
`7
`8
`prepared by first synthesizing interferon protein in the
`increased amounts of the gene product for which DNA
`manner described and additionally permitting the cell to
`I codes in the same manner as described hereinabove.
`produce the non-proteinaceous or sugar portion of in-
`Alternatively, multiple copies of foreign genes can be
`terferon and to synthesize or assemble true interferon
`generated in and ultimately expressed by eucaryotic
`cells by transforming the eucaryotic cells with DNA
`therefrom. The interferon so prepared could then be
`molecules, each of which has been formed by linking a
`recovered using conventional techniques.
`foreign DNA I to a foreign DNA II which corresponds
`In accordance with the present invention and as de-
`scribed more fully hereinafter, eucaryotic cells have
`to an amplifiable gene for a dominant selectable pheno-
`been stably transformed with precisely defined procary-
`type not normally expressed by the eucaryotic cell. The
`linkage between DNA I and DNA II is preferably in 10 otic and eucaryotic genes for which no selective criteria
`exist. The addition of a purified viral thymidine kinase
`the form of a chemical bond, particularly a bond formed
`as a result of enzymatic treatment with a ligase. Trans-
`(tk) gene to mouse cells lacking this enzyme results in
`formation with such hvbrid DNA molecules so formed
`the appearance of stable transformants which can be
`is then carried out in ~ suitable growth medium and in
`selected by their ability to grow in HAT medium. Since
`the presence of successively elevated concentrations, IS these biochemical transformants might represent a sub-
`population of competent cells which are likely to inte-
`e.g., amounts ranging from 1:1 to 10,000:1 on a molarity
`basis, of an agent which permits survival and/or identi-
`grate other unlinked genes at frequencies higher than
`fication of those eucaryotic cells which have acquired a
`the general population; con transformation experiments
`sufficiently high number of copies of the amplifiable
`were performed with the viral tk gene and bacteria-
`gene. Using this approach, eucaryotic cells which have 20 phage <I>Xl74, plasmid pBR 322 or cloned chromo-
`somal human or rabbit /3-globin gene sequences. Tk
`acquired multiple copies of the amplifiable gene for a
`dominant selectable phenotype not otherwise expressed
`transformants were cloned and analyzed for contransfer
`by the cell survive and/or are identifiable in the pres-
`of additional DNA sequences by blot hybridization. In
`ence of elevated concentrations of an agent complemen-
`this manner, mouse cell lines were identified which
`tary to the amplifiable gene which would otherwise 25 contain multiple copies of <I> X, pBR 322, or human and
`rabbit ,8-globin sequences. From one to more than 50
`result in death or inability to identify the cells.
`Although various amplifiable genes for dominant
`cotransformed sequences are integrated into high rna-
`selectable phenotypes are useful in the practices of this
`lecular weight DNA isolated from independent clones.
`invention, genes associated with drug resistance, e.g.,
`Analysis of subclones demonstrates that the contrans-
`the gene for dihydrofolatc reductase which renders 30 formed DNA is stable through many generations in
`cells resistant to methotrexate, are particularly suitable.
`culture. This cotransformation system allows the intra-
`By using either of the two approaches just described,
`duction and stable integration of virtually any defined
`multiple copies of proteinaceous or other desired mole-
`gene into cultured eucaryotic cells. Ligation to either
`viral vectors or selectable biochemical markers is not
`cules can be produced within eucaryotic cells. Thus, for
`example, multiple molecules of interferon protein, insu- 3S required.
`Cotransformation with dominant-acting markers
`lin, growth hormone, clotting factor, viral antigen or
`antibody or of interferon per se can be produced by
`should in principle permit the introduction of virtually
`eucaryotic cells, particularly mammalian cells, which
`any cloned genetic element into wild-type cultured
`have been transformed using hybrid DNA or con trans-
`eucaryotic cells. To this end, a dominant-acting, metho-
`formed using purified DNA which has been treated 40 trexate resistant, dihydrofolate reducatse gene from
`CHO A29 cells was transferred to wild-type cultured
`with calcium phosphate in the manner described herein-
`mouse cells. By demonstrating the presence of CHO
`after. Thus, this invention provides a process for pro-
`ducing highly desired, rare and costly proteinaceous
`DHFR sequences in transformants, definitive evidence
`for gene transfer was provided. Exposure of these cells
`and other biological materials in concentrations not
`4S to elevated levels of methotrexate results in enhanced
`obtainable using conventional techniques.
`Still another aspect of the present invention involves
`resistance to this drug, accompanied by amplification of
`the preparation of materials normally produced within
`the newly transferred gene. The mutant DHFR gene,
`eucaryotic cells in minute amounts such as glyco-
`therefore, has been used as a eucaryotic vector, by ligat-
`ing CHO A29 cell DNA to pBR 322 sequences prior to
`prooteins including interferon, which are in part protein
`but additionally include other chemical species such as so transformation. Amplification of the DHFR sequences
`results in amplification of the pBR sequences. The use
`sugars, ribonucleic acids, histones and the like. AI-
`though the method or methods by which cells synthe-
`of this gene as a dominant-acting vector in eucaryotic
`cells will expand the repetoire of potentially transform-
`size complicated cellular materials such as the glyco-
`proteins are poorly understood, it is anticipated that by
`able cells, no longer restricting these sort of studies to
`using the process of the present invention it will be 55 available mutants.
`possible to synthesize such materials in commercially
`Using the techniques described,the cloned chromo-
`useful quantities. Specifically, it is anticipated that after
`somal rabbit .8-globin gene has been introduced into
`inserting a gene or genes for the protein portion of a
`mouse fibroblasts by DNA-m