United States Patent [19]
`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 NY.
`The Trustees of Columbia University,
`New York, NY.
`[21] Appl.No.: 124,513
`[22] Filed:
`Feb. 25, 1980
`
`[73] Assignee:
`
`[51] Int. Cl.3 ..................... .. C12N 15/00; C12N 5/00;
`C12P 21/00; C12Q 1/68; C12Q 1/02; C12Q
`1/04
`[52] US. Cl. ...................................... .. 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 3/1974 Goore ..... ..
`4,195,125 3/1980 Wacker ........................... 5.1435/172
`
`FOREIGN PATENT DOCUMENTS
`
`2010847 7/1979 United Kingdom ...........
`
`435/71
`
`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
`.-UCLA Symposia, R. Axel and T. Maniatis, Editors,
`Academic Press, 457-475 (1979).
`'’
`Wigler et al., PNAS 76(3), 1373-1376 (1979).
`Wold et al. , PNAS 76(11), 5684-5688 (1979),
`The Merck Index, 8th Edition, Merck & Co., Inc., Rah
`way, N.J., 568 (1968).
`Szybalska et al., PNAS 48, 2026-2034 (1962).
`
`[11]
`[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., Molec. Gen. Genet, 170, 179-185
`(1979).
`Graf et al., Somatic Cell Genetics 5, 1031-1044 (1979).
`Pellicer et al., Cell 14, 131-141 (1978).
`Mercola et al., Science 208, 1033-1035 (May 1980).
`Wahl et al., J. Biol. Chem., 254(17), 8679-8689 (Sep. 10,
`1979).
`.
`Anderson et al., Scienti?c 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-Esther 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
`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
`rials by cotransformation with the desired genes and
`with ampli?able 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
`cule including the desired gene to a DNA molecule
`which includes an ampli?able gene coding for a domi
`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 ampli?able gene
`have been inserted survive.
`
`73 Claims, 2 Drawing Figures
`
`Merck Ex. 1006, pg 103
`
`

`
`U.S. Patent Aug. 16, 1983
`
`Sheet 1 0f 2
`
`COTQANSFORMATION OF EUCARYOTIC CELLS
`
`W W
`DNA I CODING FOR
`DNA II CODING FOE
`DESIRED PRoTEINAcEous
`SELECTABLE
`MATERIAL v
`MARKER Cw‘)
`
`+
`
`_
`
`NUCLEUS
`cIIRomosoIuAI. DNA
`EucARvoTIc CELL((-,K')
`
`CYTOPLA SM
`
`COTRANSFORM IN PRESENCE
`OF SELECTIVE CRITERIA (HAT)
`
`EUCARVOTIC CELL (w)
`
`DNA II
`
`DNA I
`
`1
`
`CULTURE IN PRESENCE OF
`SELECTIVE CRITERIA (I-IAT)
`
`Q
`
`DES‘RED
`MATERIAL
`
`l RECOVERY -
`
`T1?- 1~
`
`DESIRED
`
`MATERIAL
`
`DESIRED MATERIAL
`(e3. INTERFERON, INSULIMETC.)
`
`Merck Ex. 1006, pg 104
`
`

`
`US. Patent Aug. 16, 1983
`
`Sheet 2 of2
`
`4,399,216
`
`RESCUE OF PER FROM TRANSFORMED MOUSE CELLS
`
`HWD n1 CLEAVED PER 322
`l COTRANSFORM MOUSE CELLS
`
`f
`Xha. I
`
`'——l
`Amp‘
`
`I
`
`'r
`Xba 1
`
`T512.
`
`PER 322 INTEGRATED (NMOUSE DNA
`‘ CLEAVE WITH XbcLI
`
`CIRCULARIZE WITH UGASE
`TRANSFORM E.COLl X \776
`
`MOUSE DNA [N SERT
`
`/Amp'
`
`SCHEME FOR THE RESCUE OF BACTERlAL
`PLASMIDS FROM TRANSFORMED CULTURED
`CELLS USING DOUBLE SELECTION TEQHNlQUES,
`
`Merck Ex. 1006, pg 105
`
`

`
`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
`partment of Health and Human Services.
`FIELD OF THE INVENTION
`This invention concerns the introduction and expres
`sion of genetic informational material, i.e., DNA which
`includes genes coding for proteinaceous materials and
`/or genes regulating or otherwise in?uencing the pro
`duction thereof, into eucaryotic cells, that is, cells of
`organisms classi?ed under the Superkingdom Eucary
`otes including organisms of the Plant and Animal King
`doms. Such genetic intervention is commonly referred
`to as genetic engineering and in certain aspects involves
`the use of recombinant DNA technology. The inven
`tion disclosed is to be distinguished from the introduc
`tion of DNA into organisms of the Superkingdom
`Procaryotes including particularly bacteria. This dis
`tinction is based in part upon the basic differences be
`tween eucaryotic and procaryotic cells, the former
`being characterized by true nuclei formed by nuclear
`envelopes and by meiosis and the latter being vcharacter
`ized by the absence of well-de?ned nuclei and the ab
`sence of meiosis. Moreover, at the genetic level many
`genes in eucaryotes are split by non-coding sequences
`which are not continuously colinear, whereas in proca
`ryotes, the genes are continuously colinear.
`
`30
`
`35
`
`4,399,216
`2
`formation that it was not possible to distinguish tranfor'
`mants from cells which had merely undergone sponta
`neous reversion. Again, as with procaryotic cells, fur
`ther reports of eucaryotic transformation occurred in
`the literature, but such results were oftentimes not re
`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
`in this area. As a result, studies on the transformation of
`eucaryotic‘ cells were essentially restricted to viral
`genes. Graham, F. L., et al., Cold Spring Harbor Symp.
`Quant. Biol. 39: 637-650 (1975) and McCutchen, J. H.
`and Pagano, J. S., Journal National Cancer Institute, 4]:
`351-357 (1968).
`More recently, however, eucaryotic cells, speci?
`cally mammalian‘cells, were transformed with foreign
`DNA coding for a selectable phenotype. Wigler, M., et
`al., Cell 11: 223-232 (1977). This work has been ex
`tended and has resulted in the present invention
`wherein it has been discovered inter alia that eucaryotic
`cells can be cotransformed to yield transformants hav
`ing foreign DNA integrated into the chromosomal
`DNA of the eucaryotic cell nucleus. Moreover, it has
`unexpectedly been discovered that such foreign DNA
`can be expressed by the cotransformants to generate
`functional proteins. In addition, by contrast with pro
`caryotic transformants, the foreign DNA is stably ex
`pressed through hundreds of generations, a result that
`may be attributable to integration of the foreign DNA
`into the chromosomal DNA.
`The present invention provides major advances over
`bacterial systems for future use in the commercial prep
`aration of proteinaceous materials particlarly proteins
`of eucaryotic origin such as interferon protein, antibod
`ies, insulin, and the like. Such advantages include the
`ability to use unaltered genes coding 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
`cules of biological signi?cance. 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
`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
`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
`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
`lin, antibodies and the like which does not require a
`detailed molecular understanding of amino acid se
`quence.
`In addition to the problem of precursors having addi
`tional amino acids which must be removed to produce
`
`60
`
`BACKGROUND OF THE INVENTION
`Although advances in the understanding of procary
`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
`forth certain developments involving procaryotes.
`In 1944, Avery reported the transformation of a pro
`caryotic cell using DNA-mediated transfer of a cellular
`gene. Avery, O. T., et al., J. Exp. Med. 79: 137—158
`(1944). Thereafter, reports of procaryotic transforma
`tion occurred in the literature. In 1975, Cohen and oth
`ers reported results involving ?rst transformation, then
`cotransformation of the procaryote Escherichia coli.
`Kretschmer, P. 1., et al., J. Bacteriology 124: 225-231
`(1975). In the experiments reported therein the authors
`disclosed the cotransformation of procaryotic cells
`using plasmid DNA, that is, extrachromosomal DNA
`which occurs naturally in many strains of Enterobac
`teriacae. In these experiments it was found that particu
`lar cells in a CaCh-treated bacterial population are
`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
`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
`ceeded substantially independently'?of those with pro
`caryotic cells. In 1962, Szybalska, E. H. and Szybalski,
`W. PNAS 48: 2026 (1962) reported the transformation
`of mamrnlian cells but with such low frequency of trans
`
`45
`
`65
`
`Merck Ex. 1006, pg 106
`
`

`
`4,399,216
`3
`active protein, important ‘biological materials may be
`modi?ed by chemical additions after synthesis and
`cleavage. Thus, v‘for example, human-produced, inter
`feron is a glycoprotein containing’ sugar molecules in
`addition to protein. If_ produced in a bacterial cell, the
`interferon lackshthe sugar molecules which are added
`when interferon is produced in a human cell. Moreover,
`proteinaceous materials produced within bacteriatnay
`include endotoxins which can cause in?ammation if the
`proteinaceous material is administered to a mammal
`without signi?cant puri?cation. By contrast, interferon
`produced in a eucaryotic cell would be free of endotox
`
`lIlS.
`
`.
`
`5
`
`20
`
`It is therefore another important object of this inven
`tion to provide a process for producing compounds
`which include both non-proteinaceous, and protein
`aceous moieties such as glycoproteins which cannot be
`produced in bacterial cell.
`SUMMARY OF THE'INVENTION
`This invention provides a process for inserting for
`eign DNA into eucaryotic cells by cotransforming the
`cells with this foreign DNA and with unlinked DNA
`which codes for proteinaceous material associated with
`a selectable phenotype not otherwise expressed by the
`cell. The cotransformation is carried out in a suitable
`medium and in the presence of selective conditions
`permitting survival and/or identi?cation of eucaryotic
`cells which have acquired the selectable phenotype.
`The process of this invention is particularly suited for
`the insertion into eucaryotic cells of DNA which codes
`for proteinaceous materials which are not associated
`with a selectable phenotype such as interferon protein,
`insulin, growth hormones clotting factors, viral anti
`gens, antibodies and certain enzymes.
`By use of the cotransformation process of the present
`invention is it possible to produce eucaryotic cells
`which synthesize desired proteinaceous and other mate
`rials and which can'be grown in culture to produce
`these materials in quantities not obtainable with conven
`tional technology.
`_
`‘
`In one embodiment of theinvention, the cotransfor
`mation process can be used to insert multiple copies of
`genes coding for desired materials into eucaryotic cells.
`Alternatively, a multiplicity of foreign DNA molecules
`corresponding to multiple copies of a desired gene can
`be inserted into eucaryotic cells by transformation with
`molecules each of which is formed by linking a foreign
`DNA molecule to a second DNA molecule correspond
`ing to an ampli?able gene for a dominant selectable
`phenotype not otherwise expressed by the cell. The
`transformation is then carried out in the presence of
`successively elevated concentrations of an agent per
`mitting survival and/or identi?cation of eucaryotic
`cells which have acquired multiple copies of the am
`.pli?able gene. This approach is particularly useful when
`the dominant selectable phenotype is resistance to a
`drug which is lethal unless multiple copies of the drug
`resistant gene are present and the agent is the drug. 4
`By inserting multiple copies of genes coding for de
`sired materials into eucaryotic cells-according to either
`of these approaches it is possible to produce eucaryotic
`cells which yield desiredrfmaterials :in‘ high concentra
`tions and which can be grown in culture to produce
`such materials in quantities not obtainable with conven
`tional technology.
`‘
`'
`
`4
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a schematic‘flow diagram illustrating the
`Cotransformation- process in accordance with the pres
`ent invention.
`FIG. 2 is a schematic flow diagram-‘illustrating a
`process for recovering foreign DNA I‘from cotrans-. .
`formed cultured cells using double selection techniques.
`DETAILED DESCRIPTION OF THE
`INVENTION
`1
`Prior to setting forth the invention, it may be helpful
`to an‘understanding thereof to set forth de?nitions of '
`certain terms to be used hereinafter.
`Transformation means the process for changing the
`genotype of a recipient cell mediated by the introduc
`tion of puri?ed DNA. Transformation is typically de
`tected by a stable and heritable change in the phenotype
`of the recipient cell that results from an alteration in
`either the biochemical or morphological properties of
`the recipent cell.
`Cotransformation means the process for carrying out
`transformations of a recipient cell with more than one
`different gene. Cotransformation includes both simulta
`neous and sequential changes in the genotype of a recip
`ient cell mediated by the introduction of DNA corre
`sponding to either unlinked or linked genes.
`Proteinaceous material means any biopolymer formed
`from amino acids.
`Genotype means the genetic constitution of an organ
`ism as distinguished from its physical appearance.
`Phenotype means the observable properties of an or
`ganism as produced by the genotype in conjunction
`with the environment.
`Selectable phenotype is a phenotype which confers
`upon an organism ability to exist under conditions
`which kill off all organisms not possessing the pheno
`type. Examples include drug resistance or the ability to
`synthesize some molecule necessary to cell metabolism
`in a given growth medium. As used herein, selectable
`phenotypes also include identi?able phenotypes such as
`the production of materials which pass from or are
`secreted by the cell and can be detected as new pheno
`types either by functional, immunologic or biochemical
`assays.
`,Interferon protein means the proteinaceous part of the
`glycoprotein interferon, that is, the portion remaining
`after removal of the sugar portion. It includes the pro
`tein portion of interferon derived from human leuko
`cyte, ?broblast or lymphoblastoid cells.
`Chromosomal DNA means the DNA normally associ
`ated with histone in the form of chromosomes residing
`in the nucleusof a eucaryotic cell.
`Transcription means the formation of a RNA chain in
`accordance with the genetic information contained in
`the DNA. -
`Translation means the process whereby the genetic
`information in an mRNA molecule directs the order of
`speci?c amino acids during protein synthesis.
`In accordance with, the present invention, foreign
`DNA I can be inserted into any eucaryotic cell by co
`transforming the cell with DNA I and 'with unlinked
`foreign DNA II which includes a gene coding for a
`selectable phenotype not expressed by the cell unless
`acquiredvv by transformation. The cotransformation is
`carried out in a ‘suitable growth medium and in the
`presence of selective conditions such that the only cells
`
`35
`
`40
`
`KI]
`
`60.
`
`Merck Ex. 1006, pg 107
`
`

`
`4,399,216
`
`5
`
`25
`
`5
`which survive or are Otherwise altered are those which
`have required the selectable phenotype. See FIG. 1.
`Althoughth'e experim'entsdiscussed hereinafter con
`cern cultured eucaryotic cells of mammalian origin
`such as human blood'cells, mouse ?broblast cells, chin
`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 ?ow
`ers. Therefore, it is to be understood that the invention
`encompasses all eucaryotic cells even though the inven
`tion may ultimately be most useful in cotransforming
`mammalian cells.
`The present invention is especially useful in connec
`tion with the insertion into eucaryotic cells of foreign
`DNA which includes genes which code for protein
`aceous materials not associated with selectable pheno
`types. Since such proteinaceous materials are character
`ized by the fact that they are not associated with a
`selectable phenotype, cells which contain DNA coding
`therefore cannot be identi?ed except by destruction of
`the transformed celland examination of its contents.
`Examples of proteinaceus materials, the genes for
`which may be inserted into and expressed by eucaryotic
`cells using the cotransformation process include inter
`feron protein, insulin, growth hormones, clotting fac
`tors, viral antigens, enzymes and antibodies.
`Although in some cases the DNA I and DNA II may
`not need to be puri?ed to obtain integration and expres
`sion, it is oftentimes preferable that the DNAs be puri
`?ed prior to use in cotransforming cells. Such puri?ca
`tion limits the possibility of spurious results due to the
`presence of contaminants and increases the probability
`that cotransformed cells can be identi?ed and stably
`cultured. Also, although not essential, it is sometimes
`desirable that DNA I and/or DNA II have been ob
`tained by restriction endonuclease cleavage of chromo
`somal donor DNAs, such as, for example, restriction
`endonuclease cleavage of eucaryotic chromosomal
`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
`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 1:1 to about
`100,000zl.
`In a preferred embodiment of the invention, the for
`eign DNA I and/or the foreign DNA 11 are attached to
`bacterial plasmid or phage DNA prior to use in cotrans
`forming eucaryotic cells. In a particularly promising
`embodiment, foreign DNA I and/or DNA II are at
`tached to phage DNA and then encapsidated in phage
`particles prior to cotransformation.
`Although any DNA II coding for a selectable pheno
`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 for; adenine phosphoribosyl transferase. In addi
`tion, a DNA II which includes a gene coding for a
`selectable phenotype associated with drug resistance,
`e.g., a mutant dihydrofolate reductase gene which ren
`ders cells resistant to methotrexate greatly extends the
`applicability of the process.
`'
`'
`'
`'
`-
`
`40
`
`45
`
`50
`
`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
`/or identi?cation on the medium. Merely by way of
`example, a suitable medium for mouse ?broblast cells
`which have aquired the thymidine kinase gene is HAT
`described more fully hereinafter. Also, the cotransfor
`mation is carried out in the presence of selective condi
`tions which permit survival and/0r identi?cation of
`those cells which have acquired the selectable pheno
`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
`rial recovered therefrom.
`Although the desired proteinaceous materials identi
`?ed hereinabove are natural materials, the process can
`be equally useful in the production of synthetic biopoly
`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
`ties or in such impure form that their isolation and/or
`identi?cation cannot otherwise be effected. Finally, the
`invention provides a process for producing partially
`proteinaceous productslsuch 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
`DNA 1 molecules and with multiple, unlinked foreign
`DNA II molecules corresponding to multiple copies of
`an ampli?able 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
`identi?cation of cells which acquire the dominant se
`lectable phenotype. Preferably, this is done in the pres
`ence of successively higher concentrations of such an
`agent so that only those cells acquiring the highest
`number of ampli?able dominant genes (DNA II) sur
`vive and/or are identi?ed. These cells then also contain
`multiple copies of DNA I. This approach is particularly
`appropriate for the insertion of multiple copies of am
`pli?able 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
`
`Merck Ex. 1006, pg 108
`
`

`
`4,399,216
`8
`7
`prepared by ?rst 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.
`Alternatively, multiple copies of foreign genes can be
`produce the non-proteinaceous or sugar portion of in
`generated in and ultimately expressed by eucaryotic
`terferon and to synthesize or assemble true interferon
`cells by transforming the eucaryotic cells with DNA
`therefrom. The interferon so prepared could then be
`recovered using conventional techniques.
`molecules, each of which has been formed by linking a
`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 ampli?able gene for a dominant selectable pheno
`type not normally expressed by the eucaryotic cell. The
`been stably transformed with precisely de?ned procary
`linkage between DNA I and DNA II is preferably in
`otic and eucaryotic genes for which no selective criteria
`exist. The addition of a puri?ed 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 hybrid DNA molecules so formed
`the appearance of stable transformants which can be
`selected by their ability to grow in HAT medium. Since
`is then carried out in a suitable growth medium and in
`the presence of successively elevated concentrations,
`these biochemical transformants might represent a sub
`e.g., amounts ranging from 1:1 to l0,000:l on a molarity
`population of ‘ competent cells which are likely to inte
`grate other unlinked genes at frequencies higher than
`basis, of an agent which permits survival and/or identi
`the general population; contransformation experiments
`?cation of those eucaryotic cells which have acquired a
`suf?ciently high number of copies of the ampli?able
`were performed with the viral tk gene and bacterio
`gene. Using this approach, eucaryotic cells which have
`phage <l>X174, plasmid pBR 322 or cloned chromo
`acquired multiple copies of the ampli?able gene for a
`somal human or rabbit B-globin gene sequences. Tk
`dominant selectable phenotype not otherwise expressed
`transformants were cloned and analyzed for contransfer
`by the cell survive and/or are identi?able 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 identi?ed which
`tary to the ampli?able gene which would otherwise
`contain multiple copies of <I>X, pBR 322, or human and
`result in death or inability to identify the cells.
`rabbit ,B-globin sequences. From one to more than 50
`Although various ampli?able genes for dominant
`cotransformed sequences are integrated into high mo
`lecular weight DNA isolated from independent clones.
`selectable phenotypes are useful in the practices of this
`invention, genes associated with drug resistance, e.g.,
`Analysis of subclones demonstrates that the contrans
`formed DNA is stable through many generations in
`the gene for dihydrofolate reductase which renders
`culture. This cotransformation system allows the intro
`cells resistant to methotrexate, are particularly suitable.
`By using either of the two approaches just described,
`duction and stable integration of virtually any de?ned
`multiple copies of proteinaceous or other desired mole
`gene into cultured eucaryotic cells. Ligation to either
`cules can be produced within eucaryotic cells. Thus, for
`viral vectors or selectable biochemical markers is not
`required.
`example, multiple molecules of interferon protein, insu
`lin, growth hormone, clotting factor, viral antigen or
`Cotransformation with dominant-acting markers
`should in principle permit the introduction of virtually
`antibody or of interferon per se can be produced by
`eucaryotic cells, particularly mammalian cells, which
`any cloned genetic element into wild-type cultured
`have been transformed using hybrid DNA or contrans
`eucaryotic cells. To this end, a dominant-acting, metho
`formed using puri?ed DNA which has been treated
`trexate resistant, ‘ dihydrofolate reducatse gene from
`with calcium phosphate in the manner described herein
`CHO A29 cells was transferred to wild-type cultured
`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, de?nitive evidence
`and other biological materials in concentrations not
`for gene transfer was provided. Exposure of these cells
`obtainable using conventional techniques.
`to elevated levels of methotrexate results in enhanced
`resistance to this drug, accompanied by ampli?cation of
`Still another aspect of the present invention involves
`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
`prooteins including interferon, which are in part protein
`ing CHO A29 cell DNA to pBR 322 sequences prior to
`but additionally include other chemical species such as
`transformation. Ampli?cation of the DHFR sequences
`results in ampli?cation of the pBR sequences. The use
`sugars, ribonucleic acids, histones and the like. Al
`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
`available mutants.
`possible to synthesize such materials in commercially
`Using the techniques described,‘the cloned chromo
`useful quantities. Speci?cally, it is anticipated that after
`somal rabbit B-globin gene has been introduced into
`mouse ?broblasts by DNA-mediated gene transfer. The
`inserting a gene or genes for the protein portion of a
`cotransformed mouse ?broblast containing this gene
`cellular material such as a glycoprotein, which includes
`provides a unique opportunity to study the expression
`a non-protein portion, into a eucaryotic cell of the type
`which normally produces such material, the cell will
`and subsequent processing of these sequences in a heter
`ologous host. Solution hybridization experiments in
`not only produce the corresponding proteinaceous ma
`terial but will utilize already existing cellular mecha
`concert with RNA blotting techniques indicate that in
`nisms to process the proteinaceous materials, if ‘and to
`at least one transformed cell line rabbit globin sequences
`the extent necessary, and will also‘ add the appropriate
`are expressed in the cytoplasm as a polyadenylated 95
`species.