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Human growth hormone produced by recombinant dna in mouse cells

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Publication number
WO1984002534A1
WO1984002534A1 PCT/US1983/002016 US8302016W WO1984002534A1 WO 1984002534 A1 WO1984002534 A1 WO 1984002534A1 US 8302016 W US8302016 W US 8302016W WO 1984002534 A1 WO1984002534 A1 WO 1984002534A1
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Prior art keywords
cells
gene
hgh
mt
mouse
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PCT/US1983/002016
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French (fr)
Inventor
Dean H Hamer
George N Pavlakis
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Us Commerce
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/61Growth hormones [GH] (Somatotropin)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses
    • C12N2710/00011MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA Viruses dsDNA Viruses
    • C12N2710/20011Papillomaviridae
    • C12N2710/20041Use of virus, viral particle or viral elements as a vector
    • C12N2710/20043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/001Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/80Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85Vector systems having a special element relevant for transcription from vertebrates mammalian

Abstract

A recombinant DNA composed of (1) bovine papilloma virus, (2) the promoter region of the mouse metallothionein I gene and (3) human growth hormone structural sequences ligated to the metallothionein promoter was constructed. The recombinant was stably maintained as an episome and directed production of human growth hormone when introduced into cultured mammalian cells. Not only was the yield unexpectedly high, purification was vastly simplified because the growth hormone was secreted into the tissue culture medium. The process was suitable for spinner culture. Additionally, recombinant DNA molecules composed of (1) bovine papilloma virus and (2) the whole metallothionein I gene were utilized to render mouse cells resistant to toxic concentrations of cadmium. This combination was utilized as a selective marker to cotransfer other, non-selectable genes (such as human growth hormone) into mammalian cells.

Description

Human Growth Hormone Produced By Recombinant DNA

In Mouse Cells

Prior Art Statement

US Patent 4,342,832 Goeddel et al uses a synthetic gene to produce human growth hormone in bacteria.

Sarver et al, "Bovine Papilloma Virus DNA: A Novel Eu aryotic Cloning Vector," Molecular and Cellular Biology, _, No. 6, June 1981, pp 486-496 - The BPV clon- ing vector is described; the vector is -attached to pre- proinsulin gene I.

Brinster et al, "Regulation of Metallothonein- Thymidine Kinase Fusion Plasma Injected into Mouse Eggs," Nature, 296, March 1982, pp 39-42 - Metallo- thionein-I gene promoter sequences fused to herpes virus thymidine kinase are used to accomplish the expression of the kinase gene. Mouse eggs are microirijected with this piasmid.

Hairier et al, "Induction of a Mouse Metallo- thionein-I Gene in Animal Virus Vectors," Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, NY, pp. 7-12 (abstract), 1982.

Pavlakis and Hamer, "Regulation of a Metallo¬ thionein - Growth Hormone Hybrid Gene in Bovine Papilloma Virus," Proc. Natl. Acad. Sci., in press.

Mayo et al. Cell, ^i-"-^-108- MaY 1982. UCLA Symposia Abstracts; Journal of Cellular Biochemistry, Supl. 6, 1982, p. 346.

^Background of the Invention Human Growth Hormone. Growth hormone is , produced by the anterior lobe of the pituitary gland. Hypofunction of the anterior pituitary that affects the hGH production leads to hypopituitary dwarfism. Severe cases of hypopituitary dwarfism are treated with human growth hormone isolated from human cadavers. This supply is small and isolation and purification is complex and expensive. The amino acid sequence of the hGH is known, but a synthetic chemistry process is impractical.

The production of hGH in bacteria using a recombinant synthetic gene has been described (US Patent 4,342,832 Goeddel et al). The disadvantages of this process are the difficulties of separation and purifica¬ tion and the fact that the resultant product contains an amino terminal formyl-methionin residue which is not found in the normal growth hormone and could be immuno- genic in humans.

Metallothioneins. The metallothioneins are small cysteine-rich heavy metal binding proteins. They have been found in all eukaryotic species examined and in many different organs and cell types. They protect the cell from heavy metal poisoning and may also play a role in zinc and copper homeostasis.

Recently it was shown that a cloned metallo- thionein-I gene retains its inducibility by cadmium when introduced into cells by microinjection, cotransforma- tion, or transfection with simian virus 40 (SV40) recom¬ binants. The present invention constructs a metallo¬ thionein (MT)-human growth hormone (hGH) hybrid gene, clones it in a bovine papilloma virus (BPV) vector, and introduces the recombinant molecules into cultured mouse cells. The hybrid gene is regulated by cadmium, but not by dexamethasone, whereas the chromosomal genes in the same cells are induced by both agents. hGH polypeptide synthesis is inducible by cadmium in the transformed cells and very high levels of protein are accumulated.

Bovine Papilloma Viral Vectors. Bovine papil¬ loma virus DNA has been utilized as a vector to intro¬ duce various genes into mammalian cells in tissue cul¬ ture (Sarver et al., 1981).

OMPI The present invention utilizes Bovine Papil¬ loma Virus (BPV) as a vector to transfer the mouse metallothionein-I (MT) gene into mouse cells in culture. These BPV-MT recombinants direct the synthesis of large quantities of metallothionein which renders the cells resistant to cadmium (Cd) and thus act as a marker. Furthermore, Cd resistance is utilized as a dominant selection in order to co-transfer other, non-selectable genes into mammalian cells. A human growth hormone mini-gene is inserted into BPV-MT vectors and, after selection, cadmium resistant clones produce and secrete into the medium large quantities of human growth hor¬ mone. This simple dominant selection allows the intro¬ duction of non-selectable genes into many different cell types.

This vector, consisting of the 69% transform¬ ing region of the Bovine' P.apilloma Virus, as well as' a vector consisting of the whole Bovine Papilloma Virus molecule cloned in the piasmid pML2 (Sarver et al., 1982, in press; Lusky and Botchan, 1981) have been utilized in this invention to introduce human growth hormone genes into mouse cells in culture. The present invention has isolated cell lines that produce and secrete very high quantities of hGH. This is attributed to ( 1 ) the strength and inducibility of the utilized mouse metallothionein-I promoter and (2) the stability and high copy number of the recombinant DNA molecules introduced into the mouse cells.

In the first process utilized to introduce the human growth hormone gene into the cells (essentially as described by Sarver et al. , 1981), cells capable of expressing the human growth hormone gene were selected on the basis of their altered phenotype. The cells were propagated and their ability to express and secrete hGH was tested by radioimmunoassay of the tissue culture media.

The novel aspects of the present process and resulting products are that: - (1) Very large quantities of hGH are accumulated in the tissue culture medium of the transformed cells. The amount of protein is at least 100 times greater than in previously described experiments (Sarver et al. , 1981). There are two reasons for this—(a) the mouse MT-I promoter used in the present invention proved to be a strong promoter when present on a BPV vector, especially when induced by a heavy metal such as cadmium; (b) the MT-hGH hybrid gene used was found to contain sequences that stabilize the vector. The previously used vectors did not contain these sequences and were unstable. The level of .expres¬ sion of the present hybrid gene is substantially greater than could have been predicted from previous studies of this promoter (Brinster et al. , 1982; Mayo et al., 1982). (2) Some cell clones were adapted to grow in liquid culture thereby facilitating the growth of large quantities of cells. Upon introduction into liquid cul¬ ture, the ability of the clones to produce hGH was initially lowered. Therefore, daughter cell lines were selected which can grow both in liquid culture and mono- layer culture and maintain high, levels of hGH produc¬ tion.' This is the first time that it has been possible to grow cells transformed with a BPV recombinant in liquid culture and maintain satisfactory expression. (3) The hGH produced by the present procedure is pro- cessed and secreted by the mammalian host cells. There¬ fore, it is not expected to contain any extraneous amino acids at the amino terminus. This is in contrast to the hGH produced by bacterial host cells (US 4,342,832) which contains amino terminal formylmethionine. In the second process utilized, the whole mouse metallothionein gene was ligated to the BPV vector. The metallothionein gene on BPV directs the synthesis of large quantities of metallothionein which renders the cells resistant to toxic concentrations of' cadmium. Therefore, the isolation of cells that main¬ tain and express the recombinant DNA molecules is simplified: After introduction of the recombinant DNA into the cells, toxic concentrations of C Cl2 are included in the tissue culture medium. Cells that do not contain the recombinant DNA molecule are killed by Cd while the ones that express the newly introduced metallothionein gene on the BPV recombinant grow normally.

These BPV-MT vectors can be utilized to intro¬ duce other, nonselectable genes into mouse cells. For example, a human growth hormone "minigene" that does not contain any intervening sequences was inserted into such a vector, the resulting .recombinant molecules were introduced into mouse cells and cell lines resistant to Cd were isolated and propagated. Several of these cell lines tested were shown to produce and secrete high levels of the hGH encoded by the inserted gene. The novelty of this process is that it is the first time a MT gene is used as a marker for,BPV recombinants. This vector should be useful for introducing other non¬ selectable genes into cultured cells; e.g., genes for other hormones (such as insulin or calcitonin) and for virus gene products that could be used as vaccines (such as hapatitis B surface antigen).

Description of the Drawings

FIG. 1-A. The structures of the mouse

OMH metallothionein gene, the human growth hormone "minigene" and the MT-hGH hybrid gene are shown. The expected messenger RNA molecules are also shown under the genes.

FIG.1-B. The structures of recombinant viruses BPVMG6 and BPVMG7 are shown. These viruses were constructed as described in the text and were introduced into mouse C127 cells.

FIG 1-C. Piasmid BPV recombinants containing the intact mouse MT-I gene in two different orientations are shown. These molecules can be used to bring other structural sequences under the control of the MT-I pro¬ moter and to confer cadmium resistance to transformed cells. Hatched bars indicate BPV sequences, solid lines and boxes indicate MT-I sequences and wavy lines indi¬ cate. pBR322 sequences. Restriction enzyme symbols are: E, EcoRI; B, BamHI; H, Hindlll; Bg, Bglll; K, Kpnl; S, Sad; A, Avail.

FIG. 2. Construction of the MT-hGH hybrid gene and insertion into a BPV vector are shown. A 4 Kb EcoRI fragment containing the entire mouse MT-I gene (a) and a 2.1 Kb EcoRI fragment containing an hGH mini-gene (b) were inserted into a pBR322-SV40 vector. The hGH mini-gene has 3 out of 4 intervening sequences of the hGH gene removed and is functional in monkey kidney cells (see section V) . A 2 Kb Ba HI fragment, which extends from the cap site of the hGH gene to the BamHI site of pBR322, was isolated from pSVGH3C2(L) and inserted into the Bglll site in the first exon of the MT-I gene. A piasmid, pSVMTGH8, which has the hGH frag¬ ment in the same transcriptional orientation as the MT-I gene was isolated. Digestion of this piasmid with Hindlll yielded a fragment that contained 2 Kb of MT-I 5' flanking sequences and part of the first exon of the MT gene fused to the cap site of the hGH mini-gene. This fragment was inserted into the Hindlll-linearized piasmid pBPV69TD. This vector contains the 69% trans¬ forming BamHI-Hindlll fragment of Bovine Papilloma Virus-1 cloned into pBR322. rNote that the small

Hindlll-BamHI fragment of pBR322 is duplicated in this vector in order to facilitate subsequent manipulations. After cloning in JΞ. coli the four different orientations of recombinant piasmid were isolated. In two of these, designated pBPVMGδ and pBPVMG7, the pBR322 sequences can be excised by complete BamHI digestion. This generated the two molecules designated BPVMG6 and BPVMG7 in which the hybrid gene is associated with the BPV vector in both possible orientations.

FIG. 3. SV40-MT-I plasmids. The 4000 bp

EcoRI fragment containing the mouse MT-I gene was inserted into a "poison minus" pBR322 vector containing the complete SV40 genome. Solid boxes indicate MT-I structural sequences, cross-hatched boxes indicate MT-I intervening sequences, hollow boxes indicate MT-I flank¬ ing sequences, thick lines indicate SV40 sequen es and thin lines indicate pBR322 sequences. Ori, origin of SV40 DNA replication; E, direction of SV40 early tran¬ scription; L, direction of SV40 late transcription; P, PstI; B, BamHI.

FIG. 4. Bovine papilloma-metallothionein- human growth hormone recombinants are shown. Such recombinants were utilized in order to transfer a hGH mini-gene into mouse cells. These recombinants contain the 69% transforming DNA piece of BPV (hatched bars) together with complete MT gene that are derived from the vector pBPVMT5. A human growth mini-gene with no inter¬ vening sequences was ligated to this vector, together with a piece of SV40 DNA containing the origin of DNA replication (Ori). Such recombinants can replicate both

OMPI in mouse cells and in monkey cos-1 cells. B, BamHI; E, EcoRI; H, Hindlll; K, Kpnl.

FIG. 5. Bovine papilloma virus-metallo- thionein recombinants that can be utilized as vectors to transfer other, non-selectable genes into mouse cells are shown. They contain the whole BPV molecule linear¬ ized at the BamHI site (hatched bar) , the mouse metallo¬ thionein I gene and a piece of pML2 DNA that contains the pBR322 origin of replication and the penicillinase gene (wavy line). These molecules can replicate both in IS. coli (in which case they render the bacteria resistant to ampicillin) and in animal cells (in which case they render the cells resistant to heavy metals such as cadmium) . Vectors pBMTHI and pBMTHI 1 contain one and two copies of the MT gene, respectively, which is inserted into the Hindlll site of BPV viral DNA; therefore, they contain a discontinuous BPV DNA mole¬ cule. Vector pBMTK61 has convenient restriction sites for nucleases BamHI, -Sail and Sad, in which other genes can be inserted.

FIG. 6 shows the production of hGH by the BPVMG transformed cell lines. Cells were grown in 24-well plates, induced by CdCl2 or dexamethasone for 16 hrs and the hGH in the media was quantitated by radioimmunoassay. (-) uninduced cells; (C) cells induced by 1 uM CdCl2. (D) cells induced by 50nM dexa¬ methasone. Control lines (C127, ID13, NS8) gave values <1 ng/ml in this assay.

FIG. 7 shows gel transfer hybridization of total cell DNA from 12 clones containing the BPV recombinants and producing hGH. Total cell DNA was digested with: A, BamHI; B, SacI; or C, Kpnl, electro- phoresed on 1% agarose gels, blotted on nitrocellulose i filters and hybridized to a nick-translated BPV probe. Lane B is pBPVMG6 DNA digested with the same enzymes.

FIG. 8 shows gel transfer hybridization of low molecular weight Hirt supernatant DNA from transformed- cells. Line 1 is ID13 cells, a control line containing wild-type BPV. Line 2 is a clone of cells (CBMG6-9) that contains BPVMG-6 and produces hGH. The DNA was either undigested (-) or digested with nucleases Sad or Kpnl. The approximate positions of supercoiled (I), nicked circular (II) and unit-length linear molecules (III) are indicated. Notice that BPV DNA is smaller than BPVMG6 DNA. The higher bands, indicated by X on the gel, are free multimers or concatenated molecules.

FIG. 9 shows the toxicity of CdCl2 for the mouse C127 cells. Cells" were grown in 24-well plates and were treated with various concentrations of CdCl2 as indicated. .No cells survived when Cd concentrations were higher than 15 uM.

FIG. 10 shows that the transfer of the BPV-MT- hGH recombinants into mouse C127 cells renders them resistant to Cd. Mouse C127 cells were transfected with BPV-MT-hGH recombinants and subsequently treated with 20 uM CdCl2- A, C127 cells untreated; B, C127 cells treated with CdCl2 (20 uM); and C, C127 cells trans- fected with BPV-MT-hGH recombinants and treated with CdCl2 (20 uM) .

FIG. 11 shows the resistance of individual clones to Cd. Individual foci surviving in 20 uM CdCl2 were transferred and propagated. Equal numbers of cells were plated in 24-well plates and treated with (from top to bottom) 20, 40, 80, and 100 uM CdCl2« Some clones survive in 80 uM CdCl2« Clones G2-G5 con¬ tain BPV-MT-GH recombinants. Clones M3, M18 contain oven BPV-MT recombinants. Clones G4 and G5 produce and secrete large quantities of hGH.

FIG. 12 shows induction of hGH and MT pro¬ teins. Induced and uninduced cells were labeled for 1 hr with -^-^s-cys after 7 hr of induction. Cellular and media proteins were analyzed by electrophoresis on- 20% acrylamide gels and autoradiography. Figure 12-A compares media proteins from the control line ID13 (transformed with BPV-I virus) and from clone 7-4 (transformed with BPVMG7). Only 7-4 medium contains a band comigrating with authentic pituitary hGH. Figuare 12-B shows the total media proteins from cells that had been treated for 7 hr with 1 uM CdCl2 (C), 50 nM dexa- methasone (D), or no inducer (-). Figure 12-C shows the total cell proteins from the same cells analyzed in B. Notice that dexamethasone treatment inhibits overall protein synthesis but induces metallothionein pro¬ duction.

Glossary and Abbreviations MT = metallothionein

E = restriction enzyme EcoRI B = restriction enzyme BamHI H = restriction enzyme Hindlll Bg = Bglll K = restriction enzyme Kpnl S = Sad A = Avail hGH = human growth hormone BPV = bovine papilloma virus BPVgg = subgenomic BPV cleaved at BamHI site and Hindlll site Mini-gene = smaller size and missing several restriction sites of hGH genes Non-selectable genes = examples include other hormones (such as insulin or calcitonin) or viral gene products (for example, hepatitis B surface antigen) Statement of Deposit

E . coli strain HB101 (Boyer and Royland- Dussoix, J. Mol. Biol. , 41, 459, 1969), carrying piasmid PBPVMG7 has received ATCC #39242. E_. coli strain HB101 carrying piasmid pBPVMT5 has received ATCC #39239.

E . coli strain HB101 carrying piasmid pBPVMTK6 has received ATCC #39240.

]__• Goli strain HB101 carrying piasmid pBPVMTHH has received ATCC #39241.

Cell line CBMG6-9 has .received ATCC #CRL 8189.

Cell line CBMG7-4 L2 has received ATCC #CRL8187.

Cell line CBM5G5 has received ATCC #CRL8188.

Construction of Piasmid Recombinants

pBPVMG6 and pBPVMG7. The construction of

PBPVMG6 and pBPVMG7 involved three steps (see Figure 2). First, a hGH "mini-gene" was constructed. For this, the genomic sequences between the PvuII site in exon 2 and the Bglll site in exon 5 were replaced with the corre- sponding fragment of an hGH cDNA clone (Martial et al., 1979). The resulting hGH "mini-gene" is of a smaller size and it is missing several restriction sites, facilitating further manipulations. This "mini-gene" was tested for expression in monkey cells in an SV40 vector as decribed for the unaltered gene (Pavlakis et al. , 1981) and it was found that it directs the syn¬ thesis of large quantities of hGH. The "mini-gene" was propagated in E_. coli HB101 on the recombinant piasmid pSVGH3C2(L) (Figure 2). After purification, the mini-' gene was excised from the piasmid with BamHI which cuts at the 5' end of the exon 1 and in piasmid DNA down¬ stream from the gene. Second, this fragment was sub- cloned into the Bglll site in the 51 untranslated region of the mouse MT-I gene in pJYMMT(E) (Hamer and Walling, 1982) (Figure 3). This step generated a hybrid gene that consists of 1.9 Kbp of MT-I 5' flanking sequences, 68 bp of MT-I 5' untranslated sequences, the 70 bp first exon of the hGH gene, a 250 bp hGH intron, the remaining 750 bp of hGH structural sequences, and 450 bp of hGH 3' flanking sequences. Third, the hybrid gene was recovered by hindlll digestion and was inserted into pBPV69TD partially digested with Hindlll. Piasmid PBPV69TD consists of the 69% Ba HI-Hindlll transforming fragment of BPV-I cloned in pBR322 (lowy et al., 1980). The two different orientations, depicted in Figure 2, were named pBPVMG6 and pBPVMG7 and were propagated in E_. coli HB101.

Construction of BPVMG6, BPVMG7. BPVMG6 and BPVMG7 were constructed from pBPVMG6 and pBPVMG7, respectively, by excision of the pBR322 sequences with BamHI and recircularization with DNA ligase (Figure 2).

Construction of pBPVMTl, pBPVMT5. To con¬ struct recombinants pBPVMTl and pBPVMT5, piasmid ρJYMMT(E) (Fig. 3) was digested with Hindlll and the 3.3 Kb fragment containing the mouse MT-I gene and 5' flank¬ ing sequences was cloned into pBPV69TD partially digested with Hindlll. The recombinants shown in Fig. C were propagated in and isolated from E_. coli HB101.

Construction of BPVMT1 , BPVMT5. BPVMT1 and

BPVMT5 were constructed from the plasmids pBPVMTl and pBPVMT5 (Fig. 1C) by excision of the pBP.322 sequences with Hindlll.

Construction of M5G , M5G2 (Fig. 4). Two methods were utilized to construct these recombinant viruses. (1) The Hindlll linearized BPVMT5 molecule was ligated to a Hindlll fragment containing (a) an hGH mini-gene with no intervening sequences and (b) a piece

OMPI of SV40 DNA containing the SV40 origin of replication (nucleotides 5171 to 346). (2) The second method involves the construction of recombinant plasmids pBPVM5G1 and pBPVM5G2. Piasmid ρBPVMT5 was linearized by Hindlll digestion and ligated to the same hGH-SV40 fragment described above. E_. coli was transformed with the Iigation mixture and the recombinant plasmids pBPVM5G1 and pBPVM5G2 were isolated and digested with Hindlll. The resulting molecules BPVM5G1 and PBVM5G2 were circularized by DNA ligase.

Construction of pBMTK6, pBMTK61 (Fig. 5). A Kpnl fragment containing the whole mouse MT-I gene and a 1 Kb KpnI-BamHI BPV DNA piece was ligated to the Kpnl linearized piasmid pML2-BPVl. Piasmid pML2-BPVl con- tains the entire BPV1 genome cloned in the pBR322 derivative pML2 (Lusky and Botchan, 1981). The result¬ ing piasmid pBMTK6 contains the mouse MT-I gene flanked by 1 Kb directly repeated BPV DNA. It also contains a piece of pML2 DNA from the Sail site to the Hindlll site. Piasmid pBMTK61 is a derivative of pBMTK6 which is missing the directly repeated BPV DNA.

Construction of pBMTHI, pBMTHI 1 (Fig. 5). A Hindlll fragment containing the entire MT-I DNA was ligated to the Hindlll-linearized pML2-BPV1. The resulting plasmids pBMTHI and pBMTHI 1 that contain one and two MT genes respectively were propagated in an isolated from E_. coli HB101.

Construction of Mouse Cell Lines

Containing BPVMG6 and BPVMG7. Recircu- • larized BPVMG6 or BAPVMG7 molecules constructed as described above, were introduced into mouse C127 cells (Lowy et al. , 1978) by the calcium phosphate coprecipi- tation technique (Graham and Van der Eb, 1973). " After three weeks, individual foci were picked into 24-well tissue culture plates and were grown in Dulbeco's minimum essential medium with 10% fetal calf serum. The medium was tested for the presence of hGH by a specific hGH radioimmunoassay (RIA) . All 25 clones tested wer shown to produce and secrete various quantities of hGH, easily detected by RIA (Fig. 6). Cloned cell lines were named CMBG6-n (n = 1, 2, 3,...) -for those carrying the BPVMG6 recombinant and CMBG7-n (n = 1, 2, 3,...) for those carrying the BPVMG7 recombinant. To establish the status of the recombinant molecules in the transformed lines, their total DNA was extracted and analyzed by gel transfer hybridization to a BPV probe. Fig. 7 shows the results for twelve clones digested with BamHI or Sa , which cleave once, and with Kpnl, which cleaves twice. Ten of the twelve clones gave predominantly or exclu¬ sively a'single, unit-length band with BamHI and Sa , and two bands of t!he appropriate lengths with Kpnl. This demonstrates, in agreement with previous results that the recombinant molecules are maintained primarily or exclusively as episomes. By comparison with a piasmid DNA standard, it is estimated that the trans¬ formants contain between 10-100 copies/cell of the recombinant molecules. The presence of faint bands in some of the digests may reflect minor rearrangements in the DNA or the presence of more.than one cell type. Two of the clones (6-2 and 6-3) gave different restriction patterns indicative of gross rearrangements. One of these lines (6-3) was unstable and stopped producing hGH after 5 months of culture. In contrast, all of the 10 clones with the expected DNA structure continued to pro¬ duce hGH after 1 year of continuous passage. Analysis of the low molecular weight Hirt supernatant DNA (Hirt, 1969) from several clones that had been grown for 10 months showed no alterations in DNA structure and con¬ firmed the presence of supercoiled episomal molecules (Fig. 8). The presence of hGH sequences stabilizes the - - ..... - 1 5-

vector; therefore, the recombinant molecules are propa¬ gated stably in the mouse cells and produce large quan¬ tities of hGH.

Adaptation of CBMG cells to grow in spinner •■• culture. In adapting the hGH-producing cell lines to growth in spinner culture which facilitates the large- scale production of hGH- from these cells, 60 x 10^ cells were trypsinized and Innoculated into 1 It of spinner culture medium [minimum essential medium (Eagle) with modified Earle's Salts for suspension culture

(G1BC0) with 10% fetal calf serum]. After two weeks in culture, only minimal growth was detected. These cells were then subcultured at a density of 10^ cells/ml in fresh spinner culture medium. After the second passage, the cells are adapted for growth in the spinner medium with a- doubling time of approximately 40 hr. Upon introduction in the suspension culture, the ability of several clones of CBMG cells to produce hGH is dimin¬ ished. The" cells were plated from the spinner culture back onto plastic tissue culture dishes (Falcon) and screened for hGH expression by RIA of the medium. A clone designated CBMG7-4L2 was found to produce large quantities of hGH despite the alteration of the cell morphology.

Construction of mouse cells containing BPVM5,

M5G1, M5G2, pBPVMTKδ, pBPVMTHI . Recombinant molecules constructed as described above were introduced into mouse C127 cells by the calcium phosphate coprecipita- tion method (Graham and Van der Eb, 1973). 50-70% con- fluent C127 cells were treated with 20 ug of salmon sperm DNA containing 0.1-0.5 ug of recombinant DNA in 1 ml of co-precipitate per 60 mm tissue culture dish. 24 hours later the cells were trypsinized and divided into three 60 mm tissue culture dishes. Five to ten hours later the medium was changed to 5 ml of medium contain¬ ing 25 ug/ml CdCl2 (selective medium).

The selective medium was replaced every 2-3 days. Under these conditions, mouse C127 cells are killed by cadmium (Figs. 9 and 10) while transformed cells that express the inserted mouse MT-I gene become resistant (Fig. 10).

Individual foci of Cd-resistant cells were transferred into 24-well tissue culture plates and propagated. For the clones transformed with M5G1 , M5G2 the media of the propagated foci were assayed by RIA for the presence of hGH. Several clones that are cadmium resistant and express the inserted hGH "mini-gene" were isolated and named CBM5Gn (n = 1, 2, 3,...). Such clones are resistant to high concentrations of Cd (up to 80 uM, Fig.. 11).""and secrete large quantities of hGH as determined by radioimmunoassay.

hGH Production

hGH is synthesized in the pituitary as a prehormone containing a hydrophobic amino-terminal sequence that is removed during secretion. Cultured monkey kidney cells infected with SV40-hGH recombinants are capable of both processing and secreting hGH (Pavlakis et al. , Proc. Natl. Acad. Sci. USA, 78:7398- 7402, 1981). To determine if this was also true for the BPVMG transformants, cells were labeled with 35s- ysteine and the secreted proteins in the media were analyzed by gel electrophoresis. A protein co- migrating with authentic hGH was observed in the media from the BPVMG transformed cells but not from control ID13 cells (Fig. 12). Furthermore, when cells were induced for 8 hr with cadmium or dexamethasone, the amount of this protein was increased 2-fold by cadmium

OMPI but was unaffected by dexamethasone. Variable quanti¬ ties of a higher molecular weight band in the trans¬ formed cells were also observed. Parallel analysis of the labeled intracellular proteins from the same cells showed that metallothionein synthesis was induced by both cadmium and dexamethasone. Therefore, the endogenous metallothionein genes in the transformed cells have retained their responsiveness to both heavy metals and glucocorticoids. From scans of such gels it is shown that the transformed cells produce 20-60 fold more hGH than MT (MT contains 20 cystein residues, whereas hGH contains only 4).

The amount of hGH secreted by the transformed mouse cells was quantitated by radioimmunoassay. Basal levels ranged from 0.2 to 2.5 ug/ml and these levels were increased 1.3 to 2.5-fold by treatment with cadmium but not by dexamethasone.- The hGH production levels remained constant or actually increased as the cells were continuously passaged for 10 months. Measurements of cell number and media hGH for cells that had been in culture for 10 months shows basal levels of hGH produc¬ tion ranging from 2-6 x 10^ molecules/cell/day in 4 different clones.

Induction by Metals - Cadmium

The preceding section indicates that induc¬ tion for an 8-hour period of cells with cadmium has resulted in an increase of 2-fold of the amount of pro¬ tein in the system. Although cadmium was used in the experiments, other heavy metals such as zinc, copper and mercury may be utilized since it has been shown that metallothionein genes are inducible by .all the above- noted metals.

Claims

1. A method for increasing the yield of human growth hormone (hGH) consisting essentially of con¬ structing a metallothionein-human growth hormone (MT-hGH) hybrid gene, cloning said hybrid gene in a bovine papilloma virus (BPV) vector to form a recombi¬ nant piasmid, removing bacterial DNA sequences to form a MT-hGH-BPV recombinant molecule, introducing said recom¬ binant molecule into cultured mouse cells, isolating and propagating the transformed cells containing said recom¬ binant molecule, adding a non-toxic concentration of cadmium to the transformed cells, and separating and purifying the human growth hormone secreted by the transformed cells.
2. A method of increasing the yield of a non- selectable gene product consisting essentially of con- -. structing a etallothionein-non-selectable hybrid gene, cloning said hybrid gene in a bovine papilloma virus (BPV) vector to form a recombinant piasmid, removing bacterial DNA sequences to form a MT-non-selectable gene-BPV recombinant molecule, introducing said recom¬ binant molecule into cultured mouse cells, isolating and propagating transformed cells containing said recombi¬ nant molecule, adding a non-toxic concentration of cadmium to the transformed cells, and separating and purifying the product of the non—selectable gene.
3. The method of claim 1 in which the recom¬ binant piasmid is BPVMG6.
4. The method of claim 1 in which the recom- binant piasmid is pBPVMG7.
5. The method of claim 2 in which the cadmium addition is replaced by the addition of at least one metal selected from the group consisting of copper and mercury.
6. A process for the expression of non- selectable genes such as human growth hormone consisting essentially of: isolating the mouse metallothionein-1 gene and inserting the gene on a suitable first piasmid, pSVMT(E); isolating a second gene consisting of human growth hormone structural sequences and inserting said second gene on a recombinant piasmid pSVGH3C2(L) and propagating the piasmid in E. coli HB101; cleaving said second gene from pSVGH3C2 with BamHI, and subcloning said second gene into the Bglll site of th.e mouse MT-I gene in the first -piasmid to produce a hybrid gene, MT-hGH in the piasmid PSVMTGH8; cleaving piasmid pSVMTGHδ with Hindlll in order to recover said hybrid gene; inserting said hybrid gene into a suitable plasmid-bovine papilloma virus (BPV) vector, pBPV69TD; propagating said plasmid-BPV vector in E. coli HB101; removing the piasmid sequences by diges¬ tion with BamHI and recircularizing the BPV-MT-hGH recombinant molecule with DNA ligase; inserting said BPV-MT-hGH recombinant molecule in cultured mouse cells suitable for the pro¬ duction and secretion of human growth hormone in a tissue culture medium.
7. The transformed mouse cell line produced according to claim 1 and selected from one member of the group of cell lines consisting of CBMG6-9, CBMG7-4L2.
8. A process for secreting high levels of hGH consisting essentially of inserting a MT gene in a suit¬ able plasmid-BPV vector, inserting into the vector a hGH gene to form a recombinant molecule, introducing this recombinant molecule into mouse cells, treating these cells with toxic concentrations of cadmium, isolating the surviving cells, and propagating those cells which produce high levels of hGH.
9. A process for secreting high levels of a non-selectable gene product consisting essentially of inserting a MT gene in a suitable plasmid-BPV vector, inserting into the vector a non-selectable gene to form a recombinant molecule, introducing this recombinant molecule into mouse cells, treating these cells with toxic concentrations of cadmium, isolating the surviving cells, and propagating those cells which produce high levels of the non-selectable gene product.
10. A method for secreting high levels of non- selectable gene products consisting essentially of: constructing a BPV-MT recombinant piasmid vector (bovine papilloma virus-metallothionein I); inserting a non-selectable gene sequence into said BPVMT vector; introducing the BPV-MT-non-selectable gene vector into mouse cells; adding toxic concentrations of cadmium to the cells; isolating the surviving cells, and propa¬ gating those cells which produce high levels of the non- selectable gene product.
11. The method in claim 10 in which the non- selectable gene is human growth hormone (hGH) .
12. The method in claim 10 in which the cadmium addition is replaced -.by adding toxic concentra- tions of at least one metal selected from the group con¬ sisting of copper and mercury.
13. A piasmid utilized in the process of claim 10 comprising one member of the group selected from pBPVMTl, pBPVMT5, pBMTK6, pBMTK61, pBMTHI, pBMTHIT.
14. A substantially pure mouse cell line designated CBMG6-9.
15. A substantially pure mouse cell line designated CBMG7-4L2.
16. A substantially pure mouse cell line designated CBM5G5.
17. A recombinant piasmid pBPVMG6.
18. A recombinant piasmid pBPVMG7.
19. A recombinatn piasmid pBPVMT5.
20. A recombinant piasmid pBPVMTK6.
21. A recombinant piasmid pBPVMTH11.
22. The method in claim 1 in which the trans¬ formed cells are adapted to grow in spinner culture by serial passaging the cells in spinner culture, then iso¬ lating and propagating the adapted cells which produce high levels of hGH.
23. The method in claim 2 in which the trans¬ formed cells are adapted to grow in spinner culture by serial passaging the cells in spinner culture, the iso¬ lating and propagating the adapted cells which produce high levels of the non-selectable gene product.
(RE OMPI
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US4871835A (en) * 1983-07-15 1989-10-03 Bio-Technology General Corp. Analogs of hGH having antagonistic activity, and uses thereof
US5089397A (en) * 1985-02-13 1992-02-18 California Biotechnology Inc. Superior mammalian expression system
EP0217822A1 (en) * 1985-02-13 1987-04-15 Biotech Res Partners Human metallothionein-ii promoter in mammalian expression system.
EP0217822A4 (en) * 1985-02-13 1989-05-11 Biotech Res Partners Ltd Human metallothionein-ii promoter in mammalian expression system.
EP0198474A1 (en) * 1985-04-15 1986-10-22 SMITHKLINE BEECHAM BIOLOGICALS s.a. Hepatitis B surface antigen formed by recombinant DNA techniques, vaccines, diagnostics, cell lines and methods of forming same
FR2596414A1 (en) * 1986-03-28 1987-10-02 Pasteur Institut Hybridomas obtained from the lymphocytes of transgenic animals carrying a gene expressing a given protein, and method for preparing such a protein from such hybridomas
US5073490A (en) * 1986-03-28 1991-12-17 Institut Pasteur Transhybridomas
US6022543A (en) * 1987-06-22 2000-02-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6100065A (en) * 1987-06-22 2000-08-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6110706A (en) * 1987-06-22 2000-08-29 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US7709229B2 (en) * 2004-11-02 2010-05-04 Ares Trading S.A. Serum-free cell culture medium for mammalian cells
US8273553B2 (en) 2004-11-02 2012-09-25 Ares Trading S.A. Production of growth hormone in serum-free cell culture medium for mammalian cells

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