United States Patent [191
`Cohen et al.
`
`[54] PROCESS FOR PRODUCING
`BIOLOGICALLY FUNCfiONAL
`MOLECULAR CHIMERAS
`
`[75]
`
`Inventors: Stanley N. Cohen, Portola Valley;
`Herbert W. Boyer, Mill Valley, both
`of Calif.
`
`[73] Assignee: Board of Trustees of the Leland
`Stanford Jr. University, Stanford,
`Calif.
`
`[21] Appl. No.: 1,021
`
`[22] Filed:
`
`Jan. 4, 1979
`
`Related U.S. Application Data
`[63] Continuation-in-part of Ser. No. 959,288, Nov. 9, 1978,
`which is a continuation-in-part of Ser. No. 687,430,
`May 17, 1976, abandoned, which is a continuation-in(cid:173)
`part of Ser. No. 520,691, Nov. 4, 1974.
`
`Int. CJ,3 .............................................. C12P 21/00
`[51]
`[52] u.s. Cl. ······································ 435/68; 435/172;
`435/231;435/183; 435/317; 435/849;435/820;
`435/91; 435/207; 260/112.5 S; 260/27R; 435/212
`[58] Field of Search .............. 195/1, 28 N, 28 R, 112,
`195/78, 79; 435/68,172,231,183
`
`[56]
`
`3,813,316
`
`References Cited
`U.S. PATENT DOCUMENTS
`5/1974 Chakrabarty ...................... 195/28 R
`
`OTHER PUBLICATIONS
`Morrow et a!., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`3365-3369, Nov. 1972.
`Morrow et al., Proc. Nat. Acad. Sci. USA, vol. 71, pp.
`1743-1747, May 1974.
`Hershfield et al., Proc. Nat. Acad. Sci. USA, vol. 71,
`pp. 3455 et seq. (1974).
`Jackson et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`2904-2909, Oct. 1972.
`
`[II]
`
`[45]
`
`4,237,224
`Dec. 2, 1980
`
`Mertz et al., Proc. Nat. Acad. Sci. USA, vol. 69, pp.
`3370-3374, Nov. 1972.
`Cohen, et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.
`1293-1297, May 1973.
`Cohen et al., Proc. Nat. Acad. Sci. USA, vol. 70, pp.
`3240-3244, Nov. 1973.
`Chang et al., Proc. Nat. Acad. Sci, USA, vol. 71, pp.
`1030-1034, Apr. 1974.
`Ullrich et al., Science vol. 196, pp. 1313-1319, Jun.
`1977.
`Singer et al., Science vol. 181, p. 1114 (1973).
`Itakura et al., Science vol. 198, pp. 1056-1063 Dec.
`1977.
`Komaroff et al., Proc. Nat. Acad. Sci. USA, vol. 75, pp.
`3727-3731, Aug. 1978.
`Chemical and Engineering News, p. 4, May 30, 1977.
`Chemical and Engineering News, p. 6, Sep. 11, 1978.
`Primary Examiner-Alvin E Tanenholtz
`Attorney, Agent, or Firm-Bertram I. Rowland
`ABSTRACf
`[57]
`Method and compositions are provided for replication
`and expression of exogenous genes in microorganisms.
`Plasmids or virus DNA are cleaved to provide linear
`DNA having ligatable termini to which is inserted a
`gene having complementary termini, to provide a bio(cid:173)
`logically functional replicon with a desired phenotypi(cid:173)
`cal property. The replicon is inserted into a microor(cid:173)
`ganism cell by transformation. Isolation of the transfor(cid:173)
`mants provides cells for replication and expression of
`the DNA molecules present in the modified plasmid.
`The method provides a convenient and efficient way to
`introduce genetic capability into microorganisms for
`the production of nucleic acids and proteins, such as
`medically or commercially useful enzymes, which may
`have direct usefulness, or may find expression in the
`production of drugs, such as hormones, antibiotics, or
`the like, fixation of nitrogen, fermentation, utilization of
`specific feedstocks, or the like.
`
`14 Claims, No Drawings
`
`Sanofi/Regeneron Ex. 1 005, pg 82
`
`Merck Ex. 1005, pg 82
`
`

`
`•
`
`4,237,224
`
`PROCESS FOR PRODUCING BIOLOGICALLY
`. ~CTIONAL MOLECULAR CHIMERAS
`··-:":..I ,o
`The invention was supported by generous ·~iants of 5
`·
`NIH, NSF and the American Cancer Society.
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This application is a continuatin"in-part of app)icatin
`Ser. No. 959,288, ftled.Nov. 9, 1978, which is a continu~
`ation of application Ser. No; 687,430 filed May 17, 1976,
`now abandoned, which was a continuation-in-part of
`application Ser. No. 520,691, filed Nov. 4, 1974, now
`abandoned.
`
`2
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS .
`The process of this invention employs novel plas(cid:173)
`mids, which are formed by inserting DNAhaving one
`or more intact genes into a plasmid in such a location as
`to permit retention of an intact replicator locus and
`system (replicon) to provide a recombinant plasmid
`molecule. The recombinant plasmid molecule will be
`10 referred to as a "hybrid" plasmid or plasmid "chimera."
`The plasmid chimera contains genes that are capable of
`expressing at least· one phenotypical property. The
`plasmmid chimera is used to transform a susceptible and
`competent microorganism under conditions where
`15 transformation occurs. The microorganism is then
`grown under conditions which allow for separation and
`harvesting of transformants that contain the plasmid
`chimera.
`The process of this invention will be divided into the
`following stages:
`I. preparation of the recombinant plasmid or plasmid
`chimera;
`·
`II. transformation or preparation of transformants;
`and
`III. replication and transcription of the recombinant
`plasmid in transformed bacteria.
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`Although transfer of plasmids among strains of E. coli
`and other Enterobacteriaceae has long been accom- 20
`plished by conjugation and/or transduction, it has not
`been previously possible to selectively introduce partic(cid:173)
`ular species of plasmid DNA into these bacterial hosts
`or other microorganisms. Since microorganisms that
`have been transformed with plasmid DNA contain au- 25
`tonomously replicating extrachromosomal DNA spe(cid:173)
`cies having the genetic and molecular characteristics of
`the parent plasmid, transformation has enabled the se(cid:173)
`lective cloning and amplification of particular plasmid
`genes.
`··
`.,
`The ability of genes derived from totally different
`biological classes to replicate and be expressed in a
`particular microorganism permits the attainment of
`interspecies genetic recombination. Thus, it becomes
`practical to introduce into a particular microorganism, 35
`genes specifying such metabolic or synthetic functions
`as nitrogen fixation, photosynthesis, antibiotic produc(cid:173)
`tion, hormone synthesis, protein synthesis, e.g. enzymes
`or antibodies, or. the like...,-functions which are indige(cid:173)
`nous to other classes of organisms-by linking the for- 40
`eign genes to a particular plasmid or viral replicon.
`
`30
`
`BRIEF DESCRIPTION OF THE PRIOR ART
`References which.relate to the subject invention are
`Cohen, et al., Proc. Nat. Acad, Sci., USA, 69, 2110 45
`(1972); ibid, 70, 1293 (1973); ibid, 70, 3240 (1973); ibid,
`71, 1030 (1974); Morrow, et al., Proc. Nat. Acad. Sci.,
`71, 1743 (1974); Novick, Bacteriological Rev., 33, 210
`(1969); and Hershfeld, et al., Proc. Nat. Acad. Sci., in
`press; Jackson, et al., ibid, .69, 2904 (1972);
`
`Preparation of Plasmid Chimera
`In order to prepare the plasmid chimera, it is neces-
`sary to have a DNA vector, such as a plasmid or phage,
`which can be cleaved to provide an intact replicator
`locus and system (replicon), where the linear segment
`has ligatable termini or is capable of being modified to
`introduce ligatable termini. Of particular interest are
`those plasmids which have a phenotypical property,
`which allow for ready separation of transformants from
`the parent microorganism. The plasmid will be capable
`of replicating in a microorganism, particularly a bacte-
`rium which is susceptible to transformation. Various
`unicellular microorganisms can be transformed, such as
`bacteria, fungii and algae. That is, those unicellular
`organisms which are capable of being grown in cultures
`of fermentation. Since bacteria are for the most part the
`most convenient organisms to work with, bacteria will
`be hereinafter referred to as exemplary of the other
`unicellular organisms. Bacteria, which are susceptible
`to transformation, include members of the Enterobacte(cid:173)
`riaceae, such as strains of Escherichia coli; Salmonella;
`so Bacillaceae, such as Bacillus subtilis; Pneumococcus;
`Streptococcus, and Haemophilus injluenzae.
`SUMMARY OF THE INVENTION
`A wide variety of plasmids may be employed of
`greatly varying molecular weight. Normally, the plas-
`Methods and compositions are provided for geneti-
`cally transforming microorganisms, particularly bac- . mids employed will have molecular weights in the
`teria, to provide diveJ,"se genotypical capability and 55 range of about 1 X 106 to 50 X 1Q6d, more usually from
`about 1 to 20X J06d, and preferably, from about 1 to
`producing recombinant plasmids. A plasmid or viral
`lOX 106d. The desirable plasmid size is determined by a
`DNA Is modified to form a linear segment having liga-
`table termini which is joined to DNA having at least
`number of factors. First, the plasmid must be able to
`accommodate a replica tor locus and one or more genes
`one intact gene and complementary ligatable ·termini.
`The termini are then bound together to form a "hybrid" 60. that are capable of allowing replication of the plasmid.
`plasmid molecule \\;'hich is used to transform susceptible
`Secondly, the plasmid should be of a size which. pro-
`vides for a reasonable probability of recirculatization
`and compatible microorganisms. After transformation,
`with the foreign gene(s).to form the recombinant plas-
`the cells are grown and ·the transformants harvested.
`The newly functionalized microorganisms may then be
`mid chimera. Desirably, a restriction enzyme should be
`employed to carry out their new function; for example, 65 available, which will cleave the plasmid without inacti-
`by producing proteins which are the desired end prod-.
`vating the replicator locus and system associated with
`uct, or metabolities of enzymic conversion, or be lysed
`the replicator locus. Also, means must be provided for
`and the desired nucleic a~ds or proteins recovered.
`providi1,1g ligatable termini for the plasmid, which are
`
`Sanofi/Regeneron Ex. 1 005, pg 83
`
`Merck Ex. 1005, pg 83
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`

`
`4,237,224
`
`3
`complementary to the termini of the foreign gene(s) to
`allow fusion of the two DNA segments.
`Another consideration for the recombinant plasmid is
`that it be compatible with the bacterium to be trans(cid:173)
`formed. Therefore, the original plasmid will usually be S
`derived from a member of the family to which the bac(cid:173)
`terium belongs.
`The original plasmid should desirably have a pheno(cid:173)
`typical property which allows for the separation of
`transformant bacteria from parent bacteria. Particularly 10
`useful is a gene, which provides for survival selection.
`Survival selection can be achieved by providing resis(cid:173)
`tance to a growth inhibiting substance or providing a
`growth factor capability to a bacterium deficient in such
`capability.
`Conveniently, genes are available, which provide for
`antibiotic or heavy metal resistance or polypeptide re(cid:173)
`sistance, e.g. colicin. Therefore, by growing the bac(cid:173)
`teria on a medium containing a bac~riostatic or bacteri(cid:173)
`ocidal substance, such as an antibiotic, only the transfor- 20
`mants having the antibiotic resistance will survive. Il(cid:173)
`lustrative antibiotics include tetracycline, streptomycin,
`sulfa drugs, such as sulfonamide, kanamycin, neomycin,
`penicillin, chloramphenicol, or the like.
`Growth factors include the synthesis of amino acids, 2!1
`the isomerization of substrates to forms which can be
`metabolized or the like. By growing the bacteria on a
`medium which lacks the appropriate growth factor,
`only the bacteria which have been transformed and
`have the growth factor capability will clone.
`One plasmid of interest derived from E. coli is re(cid:173)
`ferred to as pSClOl and is described in Cohen, et a!.,
`Proc. Nat. Acad. Sci., USA, 70, 1293 (1972), (referred
`to in that article as Tc6-5). Further description of this
`particular plasmid and its use is found in the other arti- 3!1
`cles previously referred to.
`The plasmid pSCIOI has a molecular weight of about
`5.8X 106d and provides tetracycline resistance.
`Another plasmid of interest is colicinogenic factor EI
`(ColE!), which has a molecular weight of 4.2X JQ6d, -40
`and is also derived from E. coli. The plasmid has a single
`EcoRI substrate site and provides immunity to colicin
`El.
`In preparing the plasmid for joining with the exoge(cid:173)
`nous gene, a wide variety of techniques can be pro- -45
`vided, including the formation of or introduction of
`cohesive termini. Flush ends can be joined. Alterna(cid:173)
`tively, the plasmid and gene may be cleaved in such a
`manner that the two chains are cleaved at different sites
`to leave extensions at each end which serve as cohesive
`termini. Cohesive termini may also be introduced by
`removing nucleic acids from the opposite ends of the
`two chains or alternatively, introducing nucleic acids at
`opposite ends of the two chains.
`To illustrate, a plasmid can be cleaved with a restric- !1!1
`tion endonuclease or other DNA cleaving enzyme. The
`restriction enzyme can provide square ends, which are
`then modified to provide cohesive termini or can cleave
`in a staggered manner at different, but adjacent, sites on
`the two strands, so as to provide cohesive termini di- 60
`reedy.
`Where square ends are formed such as, for example,
`by HIN (Haemophilus influenzae RII) or pancreatic
`DNAse, one can ligate the square ends or alternatively
`one can modify the square ends by chewing back, add- 6!1
`ing particular nucleic acids, or a combination of the
`two. For example, one can employ appropriate transfer(cid:173)
`ases to add a nucleic acid to the 5' and 3' ends of the
`
`4
`DNA. Alternatively, one can chew back with an en(cid:173)
`zyme, such as a A.-exonuclease, and it is found that there
`is a high probability that cohesive termini will be
`achieved in this manner.
`An alternative way to achieve a linear segment of the
`plasmid with cohesive termini is to employ an endonu(cid:173)
`clease such as EcoRI. The endonuclease cleaves the
`two strands at different adjacent sites providing cohe(cid:173)
`sive termini directly.
`With flush ended molecules, a T41igase may be em-
`ployed for linking the termini. See, for example, Scara(cid:173)
`mella and Khorana, J. Mol. Bioi. 72: 427-444 (1972) and
`Scaramella, DNAS 69: 3389 (1972), whose disclosure is
`incorporated herein by reference.
`Another way to provide ligatable termini is to leave
`employing DNAse and Mn++ as reported by Lai and
`Nathans, J. Mol. Bioi, 89: 179 (197S).
`The plasmid, which has the replicator locus, and
`serves as the vehicle for introduction of a foreign gene
`into the bacterial cell, will hereafter be referred to as
`"the plasmid vehicle."
`It is not necessary to use plasmid, but any molecule
`capable of replication in bacteria can be employed.
`Therefore, instead of plasmid, viruses may be em(cid:173)
`ployed, which will be treated in substantially the same
`manner as the plasmid, to provide the 1igatable termini
`for joining to the foreign gene.
`If production of cohesive termini is by restriction
`endonuclease cleavage, the DNA containing the for-
`30 eign gene(s) to be bound to the plasmid vehicle will be
`cleaved in the same manner as the plasmid vehicle. If
`the cohesive termini are produced by a different tech(cid:173)
`nique, an analogous technique will noimally be em-
`ployed with the foreign gene. (By foreign gene is in(cid:173)
`tended a gene derived from a source other than the
`transformant strain.) In this way, the foreign gene(s)
`will have ligatable termini, so as to be able to covalently
`bonded to the termini of the plasmid vehicle. One can
`carry out the cleavage or digest of the plasmids to(cid:173)
`gether in the same medium or separately, combine the
`plasmids and recircularize the plasmids to form the
`plasmid chimera in the absence of active restriction
`enzyme capable of cleaving the plasmids.
`Descriptions of methods of cleavage with restriction
`enzymes may. be found in the following articles:
`Greene, et al., Methods in Moleatlar Bwlogy,'Vol. 9, ed.
`Wiclcner, R.. B., (Marcel Dekker, Inc., New York),
`"DNA Replication and Biosynthesis"; Mertz and Da(cid:173)
`vis, 69, Proc. Nat. Acad. Sci., USA, 69, 3370 (1972);
`The cleavage and non-covalent joining of the plasmid
`vehicle and the foreign DNA can be readily carried out
`with a restriction endonuclease, with the plasmid vehi(cid:173)
`cle and foreign DNA in the same or different vessels.
`Depending on the number of fragments, which are
`obtained from the DNA endonuclease digestion, as well
`as the genetic properties of the various fragmenta, diges-
`tion of the foreign DNA may be carried out separately
`and the fragments separated by centrifugation in an
`appropriate gradient. Where the desired DNA fragment
`has a phenotypical property, which allows for the ready
`isolation of its transformant, a separation step can usu-
`ally be avoided.
`Endonuclease digestion will normally be carried out
`at moderate temperatures, normally in the range of 10"
`to 40" C. in an appropriately buffered aqueous medium,
`usually at a pH of about 6.S to 8.S. Weight percent of
`total DNA in the reaction mixture will generally be
`about 1 to 20 weight percent. Time for the reaction will
`
`1!1
`
`!10
`
`Sanofi/Regeneron Ex. 1 005, pg 84
`
`Merck Ex. 1005, pg 84
`
`

`
`4,237,224
`
`20
`
`5
`vary, generaly being from 0.1 to 2 hours. The amount of
`endonuclease employed is normally in excess of that
`required, normally being from about 1 to 5 units per 10
`p.g of DNA.
`Where cleavage into a plurality of DNA fragments 5
`results, the course of the reaction can be readily fol(cid:173)
`lowed by electrophoresis. Once the digestion has gone
`to the desired degree, the endonuclease is inactivated by
`heating above about 60• C. for five minutes. The diges(cid:173)
`tion mixture may be worked up by dialysis, gradient 10
`separation, or the like; or used directly.
`After preparation of the two double stranded DNA
`sequences, the foreign gene and vector are combined
`for annealing and/or ligation to provide for a functional
`recombinant DNA structure. With plasniids, the an- 15
`nealing involves the hydrogen bonding together of the
`cohesive ends of the vector and the foreign gene to
`form a circular plasmid which has cleavage sites. The
`cleavage sites are then normally ligated to form the
`complete closed and circularized plasmid.
`The annealing, and as appropriate, recircularization
`can be performed in whole or in part in vitro or in vivo.
`Preferably, the annealing is performed in vitro. The
`annealing requires an appropriate buffered medium
`containing the DNA fragments. The temperature em- 25
`ployed initially for annealing will be about 40• to 70" C.,
`followed by a period at lower temperature, generaly
`from about to• to 30• C. The molar ratio of the two
`segments will generally be in the range of about 1-5:(cid:173)
`s:..J. The particular temperature for annealing will de- 3o
`pend upon the binding strength of the cohesive termi.
`While 0.5 hr to 2 or more days may be employed for
`annealing, it is believed that a period of0.5 to 6 his may
`be sufficient. The time employed for the annealing will
`vary with the temperature employed, the nature of the 35
`salt solution, as well as the nature of the cohesive ter-
`mini.
`The ligation, when in vitro, can be achieved in con(cid:173)
`ventional ways employing DNA ligase. Ligation is
`conveniently carried out in an aqueous solution (pH 40
`6-8) at temperatures in the range of about 5° to 40° c.
`The concentration of the DNA will generally be from
`about 10 to 100 g/ml. A sufficient amount of the DNA
`ligase or other ligating agent e.g. T 4ligase, is employed
`to provide a convenient rate of reaction, generally rang- 45
`ing from about 5 to 50 U/ml. A small amount of a pro(cid:173)
`tein e.g. albumin, may be added at concentrations of
`about 10 to 200 g!m!. The ligation with DNA ligase is
`carried out in the presence of magnesium at about 1-10
`mM.
`At the completion of the annealing or ligation, the
`solution may be chilled and is ready for use in transfor(cid:173)
`mation.
`It is not necessary to ligate the recircularized plasmid.
`prior to transformation, since it is found that this func- 55
`tion can be performed by the bacterial host. However,
`in some situations ligation prior to transformation may
`be desirable.
`The foreign DNA can be derived from a wide variety
`of sources. The DNA may be derived from eukaryotic 60
`or prokaryotic cells, viruses, and bacteriophage. The
`fragments employed will generally have molecular
`weights in the range of about 0.5 to 20 X 106d, usually in
`the range of 1 to lOX J06d. The DNA fragment may
`include one or more genes or one or more operons.
`Desirably, if the plasmid vehicle does not have a
`phenotypical property which allows for isolation of the
`transformants, the foreign DNA fragment should have
`
`6
`such property. Also, an intact promoter and base se(cid:173)
`quences coding for initiation and termination sites
`should be present for gene expression.
`In accordance with the subject invention, plasmids
`may be prepared which have replicons and genes which
`could be present in bacteria as a result of normal mating
`of bacteria. However, the subject invention provides a
`technique, whereby a replicon and gene can coexist in a
`plasmid, which is capable of being introduced into a
`unicellular organism, which could not exist in nature.
`The first type of plasmid which cannot exist in nature is
`a plasmid which derives its replicon from one organism
`and the exogenous gene from another organism, where
`the two organisms do not exchange genetic informa(cid:173)
`tion. In this situation, the two organisms will either be
`eukaryotic or prokaryotic. Those organisms which are
`able to exchange genetic information by mating are well
`known. Thus, prior to this invention, plasmids having a
`replicon and one or more genes from two sources
`which do not exchange genetic information would not
`have existed in nature. This is true, even in the event of
`mutations, and induced combinations of genes from
`different strains of the same species. For the natural
`formation of plasmids formed from a replicon and genes
`from different microorganisms it is necessary that the
`microorganisms be capable of mating and exchanging
`genetic information.
`In the situation, where the replicon comes from a
`eukaryotic or prokaryotic cell, and at least one gene
`comes from the other type of cell, this plasmid hereto(cid:173)
`fore could not have existed in nature. Thus, the subject
`invention provides new plasmids which cannot natu(cid:173)
`rally occur and can be used for transformation of unicel-
`lular organisms to introduce genes from other unicellu(cid:173)
`lar organisms, where the replicon and gene could not
`previously naturally coexist in a plasmid.
`Besides naturally occurring genes, it is feasible to
`provide synthetic genes, where fragments of DNA may
`be joined by various techniques known in the art. Thus,
`the exogenous gene may be obtained from natural
`sources" or from synthetic sources.
`The plasmid chimera contains a replicon ·which is
`compatible with a bacterium susceptible of transforma(cid:173)
`tion and at least one foreign gene which is directly or
`indirectly bonded through deoxynucleotides to the re-
`plicon to form the circularized plasmid structure. As
`indicated previously, the foreign gene normally pro(cid:173)
`vides a phenotypical property, which is absent in the
`50 parent bacterium. The foreign gene may come from
`another bacterial strain, species or family, or from a
`plant or animal cell. The original plasmid chimera will
`have been formed by in vitro covalent bonding between
`the replicon and foreign gene. Once the originally
`formed plasmid chimera has been used to prepare trans(cid:173)
`formants, the plasmid chimera will be replicated by the
`bacterial cell and cloned in vivo by growing the bac(cid:173)
`teria in an appropriate growth medium. The bacterial
`cells may be lysed and the DNA isolated by conven(cid:173)
`tional means or the bacteria continually reproduced and
`allowed to express the genotypical property of the for-
`eign DNA.
`Once a bacterium has been transformed, it is no
`longer necessary to repeat the in vitro preparation of
`65 the plasmid chimera or isolate the plasmid chimera from
`the transformant progeny. Bacterial cells can be repeat(cid:173)
`edly multiplied which v.:ill express the genotypical
`property of the foreign gene.
`
`Sanofi/Regeneron Ex. 1 005, pg 85
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`
`4,237,224
`
`8
`An alternative transformation technique may be
`found in Lederberg and Cohen, I. Bacteriol., ll9, 1072
`(1974), whose disclosure is incorporated herein by ref(cid:173)
`erence.
`
`7
`One method of distinguishing between a plasmid
`which originates in vivo from a plasmid chimera which
`originates in vitro is the formation of homoduplexes
`between an in vitro prepared plasmid chimera and the
`plasmid formed in vivo. It will be an extremely rare S
`event where a plasmid which originates in vivo will be
`the same as a plasmid chimera and will form homodu(cid:173)
`plexes with plasmid chimeras. For a discussion of
`homoduplexes, see Sharp, Cohen and Davidson, J. Mol.
`Bioi., 75, 235 (1973), and Sharp, et al, ibid, 71, 471
`(1972).
`The plasmid derived from molecular cloning need
`not homoduplex with the in vitro plasmid originally
`employed for transformation of the bacterium. The
`bacterium may carry out modification processes, which IS
`will not affect the portion of the replicon introduced
`which is necessary for replication nor the portion of the
`exogenous DNA which contains the gene providing the
`genotypical trait. Thus, nucleotides may be introduced
`or excised and, in accordance with naturally occurring 20
`mating and transduction, additional genes may be intro(cid:173)
`duced. In addition, for one or more reasons, the plas(cid:173)
`mids may be modified in vitro by techniques which are.
`known in the art. However, the plasmids obtained by 25
`molecular cloning will homoduplex as to those parts
`which relate to the original replicon and the exogenous
`gene.
`
`III. Replication and Transcription of the Plasmid
`The bacterial cells, which are employed, will be of
`such species as to allow replication of the plasmid vehi(cid:173)
`cle. A number of different bacteria which can be em-
`10 ployed, have been indicated previously. Strains which
`lack indigenous modification and restriction enzymes
`are particularly desirable for the cloning of DNA de(cid:173)
`rived from foreign sources.
`The transformation of the bacterial cells will result in
`a mixture of bacterial cells, the dominant proportion of
`which will not be transformed. or the fraction of cells
`which are transformed, some significant proportion, but
`normally a minor proportion, will have been trans(cid:173)
`formed by recombinant plasmid. Therefore, only a very
`small fraction of the total number of cells which are
`present will have the desired phenotypical characteris-
`tics.
`In order to enhance the ability to separate the desired
`bacterial clones, the bacterial cells, which have beeen
`subjected to transformation, will first be grown in a
`solution medium, so as to amplify the absolute number
`of the desired cells. The bacterial cells may then be
`harvested and streaked on an appropriate agar medium.
`Where the recombinant plasmid has a phenotype,
`which allows for ready separation of the transformed
`cells from the parent cells, this will aid in the ready
`separation of the two types of cells. As previously indi-
`cated, where the genotype provides resistance to a
`growth inhibiting material, such as an antibiotic or
`heavy metal, the cells can be grown on an agar medium
`containing the growth inhibiting substance. Only avail-
`able cells having the resistant genotype will survive. If
`the foreign gene does not provide a phenotypical prop(cid:173)
`erty, which allows for distinction between the cells
`transformed by the plasmid vehicle and the cells trans(cid:173)
`formed by the plasmid chimera, a further step is neces-
`sary to isolate the replicated plasmid chimera from tbe
`replicated plasmid vehicle. The steps include lysing of
`the cells and isolation and separation of the DNA by
`conventional means or random selection of transformed
`bacteria and characterization of DNA from'such trans-
`formants to determine which cells contain molecular
`chimeras. This is accomplished by physically character(cid:173)
`izing the DNA by electrophoresis, gradient centrifuga(cid:173)
`tion or electron microscopy.
`Cells from various clones may be harvested and the
`plasmid DNA isolated from these transformants. The
`plasmid DNA may then be analyzed in a variety of
`ways. One way is to treat the plasmid with an appropri(cid:173)
`ate restriction enzyme and analyze the resulting frag(cid:173)
`ments for the presence of the foreign gene. Other tech-
`niques have been indicated above.
`Once the recombinant plasmid has been replicated in
`a cell and isolated, the cells may be grown and multi(cid:173)
`plied and the recombinant plasmid employed for trans(cid:173)
`formation of the same or different bacterial strain.
`The subject process provides a technique for intro-
`ducing into a bacterial strain a foreign capabi,lity which
`is genetically mediated. A wide variety of genes may be
`employed as the foreign genes from a wide variety of
`sources. Any intact gene may be employed which can
`be bonded to the plasmid vehicle. The source of the
`gene can be other bacterial cells, mammalian cells, plant
`
`-45
`
`30
`
`II. Transformation
`After the recombinant plasmid or plasmid chimera
`has been prepared, it may then be used for the transfor(cid:173)
`mation of bacteria. It should be noted that the annealing
`and ligation process not only results in the formation of
`the recombinant plasmid, but also in the r:ecirculariza- 35
`tion of the plasmid vehicle. Therefore, a mixture is
`obtained of the original plasmid, the recombinant plas(cid:173)
`mid, and the foreign :ONA. Only the original plasmid
`and the DNA chimera consisting of the plasmid vehicle
`and linked foreign DNA will normally be capable of 40
`replication. When the mixture is employed for transfor(cid:173)
`mation of the bacteria, replication of both the plasmid
`vehicle genotype and the foreign genotype will occur
`with both genotypes being replicated in those cells
`having the ~ecombinant plasmid.
`Various techniques exist for transformation of a bac(cid:173)
`terial cell with plasmid DNA. A technique, which is
`particularly useful with Escherichia col~ is described in
`Cohen, et a!., ibid, 69, 2110 (1972). The bacterial cells
`are grown in an appropriate medium to a predetermined so
`optical density. For example, with E. coli strain C600,
`the optical density was 0.85 at 590 nm. The cells are
`concentrated by chilling, sedimentation and washing
`with a dilute salt solution. After centrifugation, the cells
`are resuspended in a calcium chloride solution at re- S5
`duced temperatures (approx. 5"-15" C.), sedimented,
`resuspended in a smaller volume of a calcium chloride
`solution and the cells combined with the DNA in an
`appropriately buffered calcium chloride solution and
`incubated at reduced temperatures. The concentration 60
`of Ca + + will generally be about O.Ql to 0.1 M. After a
`sufficient incubation period, generally from abo11t
`0.5-3.0 hours, the bacteria are subjected to a heat pulse
`generally in the range of 35" to 45" C. for a short period
`of time; namely from about 0.5 to 5 minutes. The trans- 65
`formed cells are then chilled and may be transferred to
`a growth medium, whereby the transformed cells hav(cid:173)
`ing the foreign genotype may be isolated.
`
`Sanofi/Regeneron Ex. 1 005, pg 86
`
`Merck Ex. 1005, pg 86
`
`

`
`4,237,224
`
`9
`cells, etc. The process is generally applicable to bacte(cid:173)
`rial cells capable of transformation. A plasmid must be
`available, which can be cleaved to provide a linear
`segment having ligatable termini, and an interact re(cid:173)
`plicator locus and system, preferably a system including 5
`a gene which provides a phenotypical property which
`allows for easy separation of the transformants. The·
`linear segment may then be annealed with a linear seg(cid:173)
`ment of DNA having one or more genes and the result(cid:173)
`ing recombinant plasmid employed for transformation 10
`of the bacteria.
`By introducing one or more exogeneous genes into a
`unicellular organism, the organism will be able to pro(cid:173)
`duce polypeptides and proteins ("poly(amino acids)")
`which the organism could not previously produce. In 15
`some instances the poly( amino acids) will have utility in
`themselves, while in other situations, particularly with
`enzymes, the enzymatic product(s) will either be useful
`in itself or useful to produce a desirabl