CA2045175C

CA2045175C - Production of proteins using homologous recombination

Info

Publication number: CA2045175C
Application number: CA002045175A
Authority: CA
Inventors: Arthur I. Skoultchi
Original assignee: Cell Genesys Inc
Current assignee: Cell Genesys Inc
Priority date: 1989-11-06
Filing date: 1990-11-06
Publication date: 2003-03-18
Anticipated expiration: 2010-11-06
Also published as: EP0452484A4; EP0452484B1; DE69032809D1; US5981214A; EP0452484A1; DE69027526D1; WO1991006667A1; US5578461A; US6015708A; AU635844B2; ATE139574T1; EP0747485B1; DK0452484T3; NO309238B1; AU7740791A; ES2090297T5; EP0747485A1; ES2127458T3; NO912642D0; WO1991006666A1

Abstract

Methods and compositions are provided for expression of mammalian genes in culture. An amplifiable gene is introduced by homologous recombination in juxtaposition to a target gene, the resulting combination of amplifiable gene and target gene transferred to a convenient host and the target gene amplified by means of the amplifiable gene.
The resulting expression host may then be grown in culture with enhanced expression of the target gene.

Description

wo 9iio6s~~ Pcrm~9oio6a~

i~, ; Te a PRODUCTION OF PROTEINS
USING HOMOLOGOUH RECOMBINATION
INTRODUCTION
Technical Field The field of this invention is the expression of mammalian proteins.
Background The discoveries of restriction enzymes, cloning, sequencing, reverse transcriptase, and monoclonal antibodies has resulted in extraordinary capabilities in isolating, identifying, and manipulating nucleic acid sequences. As a result of these capabilities, numerous genes and their transcriptional control elements have been identified and manipulated. The genes have been used for producing large amounts of a desired protein in heterologous halts (bacterial and eukaryotic host cell systems).
In many cases, the process of obtaining coding sequences and eliciting their expression has been a long and arduous one. The identification of the coding sequence, either cDNA or genomic DNA, has frequently involved the construction of libraries, identification of fragments of the open reading frame, examining the flanking sequences, and the like. In mammalian genes where introns are frequently encountered, in many instances, the coding region has been only small fraction of the total nucleic acid associated with the gene. In other cases, pseudogenes or multi-membered gene families have obscured the ability to isolate a particular gene of interest.
Nevertheless, as techniques have impraved, there has ~yn 9~/0~7 PCT/US90/06436 ,..

2 .l ~~ ' ° .a. y '~
been a continuous parade of successful identifications and isolation of genes of interest.
In many situations one is primarily interested in a source of the protein product. The cell type in the body which produces the protein is frequently an inadequate source, since the protein may be produced in low amounts, the protein may only be produced in a differentiated host cell which is only difficultly grown in culture, or the host cell, particularly a human cell, is not economic or efficient in a culture process far production of the product. There is, therefore, significant interest in developing alternative techniques for producing proteins of interest in culture with cells which provide for economic and efficient production of the desired protein and, when possible, appropriate processing of the protein product.
Relevant Literature Mansour et al., Nature, 336:348-352 (1988), describe a general strategy for targeting mutations to non-selectable genes. Weidle et al., Gene, 66:193--203, (1988), describe amplification of tissue-type plasminogen activator with a DHFR gene and loss of amplification in the absence of selective pressure.
Murnane and Yezzi, Somatic Cell and Molecular Genetics, 14:273-286, (1988), describe transformation of a human cell line with an integrated selectable gene marker lacking a transcriptional promoter, with tandem duplication and amplification of the gene marker. Thomas and Capecchi, Cell, 51:503-512, (19871, describe site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Song et al., Proc. Natl. Acad. Sci. USA, 84:6820-6824, (1987), describe homologous recombination in human cells by a two staged integration. Liskay et al., "Homologous Recombination Between Repeated Chromosomal Sequences in Mouse Cells," Cold Spring Harbor, Symp.

Quant. Biol. 49:13-189, (1984), describe integration of two different mutations of the same gene and homologous recombination between the mutant genes.
Rubnitz and Subramani, Mol. and Cell. Biol. _4:2253-2258, (1984), describe the minimum amount of homology required for homologous recombination in mammalian cells. Rim and Smithies, Nucl. Acids. Res. _16:8887-8903, (1988), describe an assay for homologous recombination using the polymerase chain reaction.
SUMMARY OF THE INVENTION
Expression of mammalian proteins of interest is achieved by employing homologous recombination for integration of an amplifiable gene and other regulatory sequences in proximity to a gene of interest without interruption of the production of a proper transcript. The region comprising the amplifiable gene and the gene of interest may be amplified, the genome fragmented and directly or indirectly transferred to an expression host for expression of the target protein. If not previously amplified, the target region is then amplified, and the cell population screened for cells producing the target protein. Cells which produce the target protein at high and stable levels are expanded and used for expression of the target protein.

3a This invention provides a method for producing mammalian proteins comprising:
transforn~ing mammalian host cells comprising an endogenous target gene with a construct comprising an amplifiable gene and/or a heterologous nucleotide regulatory sequence and at least one flanking region homologous to a region of the host cell genome S within or proximal to said endogenous target gene, so that the amplifiable gene and/or heterologous regulatory sequence are integrated via homologous recombination into the genome of the mammalian cells and the amplifiable gene and/or heterologous regulatory sequence become operatively associated with said endogenous target gene so that said endogenous target gene is amplified and/or so that expression of said endogenous target gene is controlled by said nucleotide regulatory sequence; selecting for cells comprising said construct by means of said amplifiable gene or other marker present in said construct; and culturing said cells comprising said construct under conditions wherein the targeted gene is expressed and the protein encoded by the targeted gene is produced.
This invention provides a method of integrating an amplifiable gene and/or a 1 S heterologous nucleotide regulatory sequence into the genome of a mammalian cell that contains an endogenous target gene comprising inserting said amplifiable gene and/or said heterologous nucleotide regulatory sequence, by targeted homologous recombination, into the genome of said mammalian cell wherein said endogenous target gene is amplified when said amplifiable gene is amplified and/or wherein expression of said endogenous target gene is controlled by said heterologous nucleotide regulatory sequence.
This invention provides a method for amplifying gene expression in a mammalian host cell having an endogenous target gene comprising: (a) transforming a mammalian host cell with an amplifiable gene, flanked by a nucleotide sequence homologous to a region of the host cell genome within or proximal to the endogenous target gene, so that the amplitiable gene is integrated by homologous recombination into the genome of the mammalian host cell; and (b) selecting a transformed mammalian host cell in which the integrated amplifiable gene is operatively associated with the endogenous target gene, so that the endogenous target gene coding sequence is not disrupted and so that the endogenous target gene is amplified and expressed when the mammalian cell is cultured under conditions that amplify the amplifiable gene.

3b This invention provides a mammalian host cell having an endogenous target gene comprising an amplifiable gene and/or heterologous nucleotide regulatory sequence, integrated by homologous recombination, into the genome of the mammalian host cell in operative association with the endogenous target gene so that said endogenous target gene is amplified when said amplifiable gene is amplified and/or so that expression of said endogenous target gene is controlled by said heterologous nucleotide regulatory sequence wherein said amplifiable gene is at other than its wild type site.
This invention provides a method for large scale production of a mammalian target gene product in cell culture, comprising: (1) culturing a mammalian continuous cell line which was prepared by the steps of: (a) integrating, via targeted homologous recombination, a nucleotide regulatory sequence heterologous to the target gene, into the genome of a mammalian host cell, so that the integrated nucleotide regulatory sequence is operably associated with the mammalian target gene contained in the host cell genome to form a recombined manunalian target gene; and (b) transferring the recombined 1 S mammalian target gene to a mammalian continuous cell line compatible with the integrated nucleotide regulatory sequence, so that the mammalian target gene product is expressed by the mammalian continuous cell line in culture; and (2) recovering the mammalian target gene product from the cell culture.
This invention provides a method for producing a mammalian continuous cell line used for large-scale protein production in culture, comprising: (a) integrating, via targeted homologous recombination, a nucleotide regulatory sequence heterologous to a mammalian target gene contained in a mammalian host cell, so that the integrated nucleotide regulatory sequence is operably associated with the mammalian target gene to form a recombined mammalian target gene; and (b) transferring the recombined ?S mammalian target gene to a mammalian continuous cell line compatible with the integrated nucleotide regulatory sequence, so that the mammalian target gene product is expressed by the mammalian continuous cell line in culture.
This invention provides a mammalian continuous cell line, the genome of which contains a heterologous genomic DNA encoding a gene product of interest operatively associated with: (a) a nucleotide regulatory sequence different from the wild-type 3c regulatory sequence normally associated with the heterologous genomic DNA; and (b) an amplifiable gene, so that expression of the heterologous genomic DNA is controlled by the regulatory sequence and is amplified when the mammalian continuous cell line is cultured under conditions that amplify the amplifiable gene.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic illustration of the plasmid pCG. l showing the sequence of the modified polylinker;
FIG. 2 is a diagrammatic illustration of the construction of the plasmid pCG.HRI;
FIG. 3 is a diagrammatic illustration of the result of targeting the EPO locus by homologous recombination with the DNA from pCG.HRI cut with NotI;

WO 91/06667 PCf/U590/06436 4 ~r~'q_ ~ i_~
FIG. 4 is a diagrammatic illustration of the PCR
amplication fragment produced from cells in which a homologous recombination event has occurred.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Methads and compositions are provided for production of mammalian proteins of interest in culture. The method employs homologous recombination in a host cell far integrating an amplifiable gene in the vicinity of a target gene, which target gene encodes the protein of interest. The region comprising both the amplifiable gene and target gene will be referred to as the amplifiable region. The resulting transformed primary cells may now be subjected to conditions which select for amplification, or the amplification may be performed subsequently. "Transform" includes transform, transfect, transduce, conjugation, fusion, electroporation or any other technique for introducing DNA into a viable cell. The chromosomes or DNA of the transformed cells are then used to transfer the amplifiable region into the genome of secondary _ expression host cells, where the target region, if not previously amplified sufficiently or at all, is further amplified. The resulting cell lines are screened far production of the target protein and secondary cell lines selected for desired levels of production, which cells may be expanded and used for production of the desired protein in culture.
The primary cell may be any mammalian cell of interest, particularly mammalian cells which do not grow readily in culture, more particularly primate cells, especially human cells, where the human cells may be normal cells, including embryonic or neoplastic cells, particularly normal cells. Various cell types may be employed as the primary cells, including fibroblasts, particularly diploid skin fibroblasts, lymphocytes, epithelial cells, neurons, endothelial WO 9!/06667 ~'CT/US90/06436 'J ~.~
cells, or other somatic cells, or germ cells. Of particular interest are skin fibroblasts, which can be readily propagated to provide for large numbers of normal cells, embryonic kidney cells, and the like.

5 These cells may or may not be expressing the gene of interest. In those instances where the target gene is inducible or only expressed in certain differentiated cells, one may select cells in which the target gene is expressed, which may require immortalized cells capable of growth in culture.
A number of amplifiable genes exist, where by appropriate use of a selection agent, a gene integrated in the genome will be amplified with adjacent flanking DNA. Amplifiable genes include dihydrofolate reductase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. The amplifiable gene will have transcriptional signals which are functional in the secondary or expression host and desirably be functional in the primary host, particularly where amplification is employed in the primary host or the amplifiable gene is used as a marker.
The target genes may be any gene of interest, there already having been a large number of proteins of interest identified and isolated with continual additions to the list. Proteins of interest include cytokines, such as interleukins 1-10;
growth factors such as EGF, FGF, PDGF, and TGF;
somatotropins; growth hormones; colony stimulating factors, such as G-, M-, and GM-CSF; erythropoietin;
plasminogen activators, such as tissue and urine;
enzymes, such as superoxide dismutase; interferons;
T-cell receptors; surface membrane proteins; insulin;
lipoproteins; ~1-antitrypsin; CD proteins, such as CD3, 4, 8, 19; clotting factors, e.g., Factor VIIIc and von Willebrands factor; anticlotting factors, such as Protein C; atrial naturetic factor, tumor necrosis W(191 /06667 PC1'/US90106436 1 r. :.:, ,~. .i ~

factor; transport proteins; homing receptars;
addressins; regulatory proteins; etc.
For homologous recombination, constructs will be prepared where the amplifiable gene will be flanked on one or bath sides with DNA homologous with the DNA of the target region. The homologous DNA will generally be within 100 kb, usually 50 kb, preferably about 25 kb, of the transcribed region of the target gene, more preferably within 2 kb of the target gene.
By gene is intended the coding region and those sequences required for transcription of a mature mI~NA. The homologous DNA may include the 5'-upstream region comprising any enhancer sequences, transcriptional initiation sequences, the region 5' of these sequences, or the like. The homologous region may include a portion of the coding region, where the coding region may be comprised only of an open reading frame or combination of axons and introns. The homologous region may comprise all or a portion of an intron, where all or a portion of one or more axons may also be present. Alternatively, the homologous region may comprise the 3'-region, so as to comprise all or a portion of the transcription termination region, or the region 3' of this region. The homologous regions may extend over all or a portion of the target gene or be outside the target gene comprising all or a portion of the transcriptional regulatory regions and/or the structural gene. For the most part, the homologous sequence will be joined to the amplifiable gene, proximally or distally.
Usually a~sequence other than the wild-type sequence normally associated with the target gene will be used to separate the homologous sequence from the amplifiable gene on at least one side of the amplifiable gene. Some portion of the sequence may be the 5' or 3' sequence associated with the amplifiable gene, as a result of the manipulations associated with the amplifiable gene.

7 ~9 ~,' ,, ,;. ~_ a,'' ~~3 The homologous regions flanking the amplifiable gene need not be identical to the target region, where _in vitro mutagenesis is desired. For example, one may wish to change the transcriptional initiation region for the target gene, so that a portion of the homologous region might comprise nucleotides different from the wild-type 5' region of the target gene. Alternatively, one could provide for insertion of a transcriptional initiation region different from the wild-type initiation region between the wild-type initiation region and the structural gene. Similarly, one might wish to introduce various mutations into the structural gene, so that the homologous region would comprise mismatches, resulting in a change in the encoded protein. For example, a signal leader sequence would be introduced in proper reading frame with the target gene to provide for secretion of the target protein expression product. Alternatively, one might change the 3' region, e.g., untranslated region, polyadenylation site, etc. of the target gene.
Therefore, by homologous recombination, one can provide for maintaining the integrity of the target gene, so as to express the wild-type protein under the transcriptional regulation of the wild-type promoter or one may provide for a change in transcriptional regulation, processing or sequence of the target gene. In some instances, one may wish to introduce an enhancer in relation to the transcriptional initiation region, which can be provided by, fox example, integration of the amplifiable gene associated with the enhancer in a region upstream from the transcriptional initiation regulatory region or in an intron or even downstream from the target gene.
In order to prepare the subject constructs,.
it will be necessary to know the sequence which is targeted for homologous recombination. While it is WO 91f06G67 P(.'T/'US90/06436 F!~ n n.,. _. ,.._, .., ,.
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reported that a sequence of 14 bases complementary to a sequence in a genome may provide for homologous recombination, normally the individual flanking sequences will be at least about 150 bp, and may be 12 kb or more, usually not more than about 8 kb. The size of the flanking regions will be determined by the size of the known sequence, the a:umber of sequences in the genome which may have homology to the site for integration, whether mutagenesis is involved and the extent of separation of the regions for mutagenesis, the particular site for integration, or the like.
The integrating constructs may be prepared in accordance with conventional ways, where sequences may be synthesized, isolated from natural sources, manipulated, cloned, ligated, subjected to in vitro mutagenesis, primer repair, or the like. At various stages, the joined sequences may be cloned, and analyzed by restriction analysis, sequencing, or the like. Usually the construct will be carried on a cloning vector comgrising a replication system functional in a prokaryotic host, e.g., E. coli, and a marker for selection, e.g., biocide resistance, complementation to an auxotrophic host, etc. Other functional sequences may also be present, such as polylinkers, for ease of introduction and excision of the construct or portions thereof, or the like. A
large number of cloning vectors are available such as pBR322, the pU0 series, etc.
Once the construct is prepared, it may then be used for homologous recombination in the primary cell target. Various techniques may be employed for integrating the construct into the genome of the primary cell without being joined to a replication system functional in the primary host. See for example, U.S. Patent No. 4,319,216, as well as the references cited in the Relevant Literature section.
Alternatively, the construct may be inserted into an WO 9i/06667 P~°T/US90/06436 c~ n ~ ~. ~ ,., ,-.
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appropriate vector, usually having a viral replication system, such as SV~O, bovine papilloma virus, adenovirus, or the like. The linear DNA sequence vector may also have a selectable marker for identifying transfected cells. Selectable markers include the neo gene, allowing for selection with 6418, the herpes _tk gene for selection with HAT
medium, get gene with mycophenolic acid, complementation of an auxotrophic host, etc.
The vector may or may not be capable of stable maintenance in the host. Where the vector is capable of stable maintenance, the cells will be screened for homologous integration of the vector into the genome of the host, where various techniques for curing the cells may be employed. Where the vector is not capable of stable maintenance, for example, where a temperature sensitive replication system is employed, one may change the temperature from the permissive temperature to the non-permissive temperature, so that the cells may be cured of the vector. Tn this case, only those cells having integration of the construct comprising the amplifiable gene and, when present, the selectable marker, will be able to survive selection.
Where a selectable marker is present, one may select for the presence of the construct by means of the selectable marker. Where the selectable marker is not present, one may select for the presence of the construct by the amplifiable gene. For the neo gene or the herpes tk gene, one could employ a medium for growth of the transformants of about 0.1-1 g/ml of 6418 or HAT medium respectively. Where DHFR is the amplifiable gene, the selective medium may include from about 0.01-0.25 ~tM of methotrexate.
In carrying out the homologous recombination, the DNA will be introduced into the primary cells. Techniques which may be used include calcium phosphate/DNA co-precipitates, microinjection WO 91/06b67 1'GT/US90/06436 I O ~ L ':.': ~' '. :~ ;.3 of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like. The DNA may be single or double stranded .DNA, linear or circular. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enz~rmoloqy (1989), Keown et al., Methods and Enzymology (1990) Vol. 185, pp. 527-537 and Mansour _et _al., Nature, 336:348-352, (1988).
I0 Upstream and/or downstream from the target region construct may be a gene which provides for identification of whether a double crossover has occurred. For this purpose, the herpes simplex virus thymidine kinase gene may be employed since the presence of the thymidine kinase gene may be detected by the use of nucleoside analogs, such as acyclovir or gancyclovir, for their cytotoxic effects on cells that contain a functional HSV-tk gene. The absence of sensitivity to these nucleoside analogs indicates the absence of the thymidine kinase and, therefore, where homologous recombination has occurred, that a double crossover event has also occurred.
The presence of the marker gene as evidenced by resistance to a biocide or growth in a medium which selects for the presence of the marker gene, establishes the presence and integration of the target construct into the host genome. No further selection need be made at this time, since the selection will be made in the secondary expression host, where expression of the amplified target gene may be detected.' If one wishes, one can determine whether homologous recombination has occurred by employing PCR
and sequencing the resulting amplified DNA sequences.
If desired, amplification may be performed at this time by stressing the primary cells with the appropriate amplifying reagent, so that mufti-copies of the target gene are obtained. Alternatively, WO 91/06667 PCf/US90106A36 11 ~ ~ ,,! w .~- y ~.~
amplification may await transfer to the secondary cell expression host.
High molecular weight DNA, greater than about 20kb, preferably greater than about 50kb DNA or preferably metaphase chromosomes are prepared from the primary recipient cell strain having the appropriate integration of the amplification vector. Preparation and isolation techniques axe described by Nelson and Housman, In Gene Transfer (ed. R. Kucherlapati) Plenum Press, 1986. The DNA may then be introduced in the same manner as described above into the secondary host expression cells, using the same or different techniques than employed for the primary cells.
Various mammalian expression hosts are available and may be employed. These hosts include CHO cells, monkey kidney cells, C127 mouse fibroblasts, 3T3 mouse cells, Vero cells, etc. Desirably the hosts will have a negative background fox the amplifiable gene or a gene which is substantially less responsive to the amplifying agent.
The transformed cells are grown in selective medium containing about 0.01-0.5 ~M methotrexate and, where another marker is present, e.g., the neo gene, the medium may contain from about 0.1-1 mg/ml 6418.
The resistant colonies are isolated and may then be analyzed for the presence of the construct in juxtaposition to the target gene. This may be as a result of detection of expression of the target gene product, where there will normally be a negative background for the target gene product, use of PCR, Southern hybridization, or the like.
The cells containing the construct are then expanded and subjected to selection and amplification with media containing progressively higher concentrations of the amplifying reagent, for example, 0.5-200 ~M of methotrexate for the DHFR gene, and may be analyzed at each selection step for production of the target product. Expansion will W~ 91/0b667 PGT/US90/06436 12 ~ ,'~ v ~~, '~ ~.3 ;~ .x . _~
include at least duplication and may result in at least 5 copies, preferably 10 copies or more in a tandem relationship. Thus protein production will be increased at least 1.5 fold from expression from a single copy, usually at least 3 fold, preferably at least 5 fold.
The various clones may then be screened for optimum stable production of the target product and these clones may then be expanded and used commercially for production in culture. In this manner, high yields of a product may be obtained, without the necessity of isolating the message and doing the various manipulations associated with genetic engineering or isolating the genomic gene, where very large genes can be a major research and development effort.
The following examples are offered by way of illustration and not by way of limitation.

Cells Normal human diploid skin fibroblasts, ("primary recipient") are propagated in EEMEM medium supplemented with 20$ fetal calf serum. Dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CH0) DUKX-Bil cells (Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA _77:9216-4220 (1980)) ("secondary recipient") are propagated in alpha-medium supplemented with 10~
dialyzed fetal bovine serum.
DNA Rector The amplification vector is constructed from pUCl9 (Yanisch-Perron et al., Gene 33:103-119 (1985)). A 1.8 kb HaeII fragment containing a hygromycin B phosphotransferase gene (hph) driven by the herpes simplex virus thymidine kinase (HSV tk) promoter is isolated from pHyg (Sugden et al., Mol.

WO 91 /06b67 PCT/ 0590/06436 y ;j ~ ~ ;~;' ,"
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Cell. Biol. _5:410-413 (1985)) by digestion with HaeII
and gel electrophoresis. Synthetic adaptors are added onto this fragment to convert the HaeII ends into H_indIII ends and the resulting fragment is joined to pUCl9 digested with HindIII. The resulting plasmid pUCH contains the hygromycin cassette such that transcription of hph and beta-lactamase are in the opposite orientation. A 1.3 kb SalI fragment containing a DHFR gene driven by SV40 transcriptional signals is isolated from pTND (Connors et al., DNA
_7:651-661 (1988)) by digestion with Sall and gel electrophoresis. This fragment is ligated to pUCH
digested with SalI. The resulting plasmid pUCD
contains the DHFR cassette such that DHFR and are transcribed in the same direction. A 1.76 kb BamHI
fragment from the phage F15 (Friezner Degen et al., J.
Biol. Chem. 261:6972-6985 (1986)) which contains 1.45 kb of DNA flanking the transcriptional start of human tissue plasminogen activator (t-PA) in addition to the first axon and part of the first intron is isolated by gel electrophoresis after _BamHI digestion. This fragment is joined to pUCD following digestion of the latter with BamHI. The resulting plasmid pUCG has the promoter of the t-PA fragment oriented opposite to that of the DHFR cassette. The t-PA fragment contains a single _Ncol site, which is not unique to pUCG. A
partial NcoI digest is carried out and a Notl linker is inserted. The resulting plasmid pCG contains a unique Notl site in the t-PA fragment which allows the plasmid to be linearized prior to transformation of the primary human diploid fibroblasts in order to increase the frequency of homologous recombination (ICucherlapati et al., Proc. Natl. Acad. Sci. USA
81:3153-3157 (1984)).
_Prep_aration of Primary Recipients The plasmid pCG linearized with NotI is introduced into the primary recipients by WO 91/06657 PCf/US90/06436 14 ~ ~~ r. ,~ .:.. z:! _.9 electroporation employing DNA at lOnM. The resulting cells are then grown in selective medium (EEMEM with 200 ,~g/ml hygromycin B). Resistant colonies are isolated and analyzed by PCR (Kim and Smithies, Nucleic Acids Res. _16:8887-8903 (1988)) using as primers the sequences GCGGCCTCGGCCTCTGCATA and CATCTCCCCTCTGGAGTGGA to distinguish homologous integrants from random ones. Amplification of cellular DNA by PCR using these two primers yields a fragment of 1.9 kb only when DNA from correctly targeted cells is present. Cells comprising the DHFR
gene integrated into the t-PA region are expanded and used as a source of genetic material for preparation of secondary recipients.
Preparation of Secondary Recipients Metaphase chromosomes are prepared Nelson et _al., J. Mol. Appl. Genet. _2:563-577 (1984)) from recipients demonstrating homologous recombination with the DHFR and are then transformed in DHFR-deficient CHO cells by calcium phosphate mediated gene transfer (Nelson et al., J. Mol. Appl. Genet. 2:563-577 (1984)).~The cells are then grown in selective medium (alpha-medium containing 200 ~g/ml hygromycin B).
Resistant colonies are isolated and analyzed for expression of human t-PA (Kaufman et al., Mol. Cell.
Biol. 5:1750-1759 (1985)). The cell clones are then grown in selective medium containing progressively higher concentrations of methotrexate (.02-80 /~M, with steps of 4-fold increases in concentration). After this amplification procedure, the cells are harvested and the human t-PA is analyzed employing an ELISA
assay with a monoclonal antibody specific for t-PA
(Weidle and Buckel, Gene 51:31-41 (1987)). Clones providing for high levels of expression of t-PA are stored for subsequent use.

f ;~. .:,l r.
15 ~, : - .. ~.' ~.~ _3 Isolation of a Genomic Clone Containing Sequences for Targeting Erythropoietin A clone was obtained by screening a human placental DNA genomic library (Clontech) in EMBL 3-SP6/T7 using two 36 by oligonucleotide probes 5'-CTGGGTTGCTGAGTTCCGCAAAGTAGCTGGGTCTGG-3' and 5'-CGGGGGTCGGGGCTGTTATCTGCATGTGTGCGTGCG-3' to the presumed promoter region of human erythropoietin.
From this clone two subclones were created in pSP72 (Krieg and Melton (1987) Meth. Enzymol. 155, 397-415), one containing a 5 kb BamHI-HindIII fragment from the region upstream to the coding region of EPO (pTD.l) and one containing a 5 kb HindIII-BamHI fragment coding for EPO (pTD.2).
Construction of DNA Fragment for Targeting Erythropoietin A plasmid pCG.l was constructed by replacement of the polylinker of pBluescript SK(-) (Stratagene) between the Sacl and Kpnl sites with a synthetic double stranded 72 base pair DNA fragment (FIG. 1). Referring to FIG. 2, into pCG.l was cloned between the HindIII and Xbal sites a 678 by fragment containing the enhancer and promoter of the immediate early gene of human cytomegalovirus (CMV, Boshart et al (1985) Cell 41, 521-530) obtained by a PCR
amplification of the plasmid pUCH.CMV (gift of M. Calos, Stanford U.) using the oligonucleotide primers 5'-CGCCAAGCTTGGCCATTGCATACGTT-3' and 5'-GAGGTCTAGACGGTTCACTAAACGAGCTCT-3' in order to engineer HindIII and Xbal sites respectively onto the ends of the resultant fragment. The resultant plasmid pCG.CMv was used for further constructions.
The 620 by BstEII-Xbal fragment from the pTD.2 was joined by the use of a BstEII-XbaI adapter to pCG.CMi~ restricted with Xbal to create the plasmid WC~ 91/06667 PCT/US90/05436 '~V r~ y) pCG.GMV/EPO, in which the BstEII site of the EPO
fragment is next to the promoter end of the CMV
fragment. Into pCG.CMV/EPO was cloned successively a 1.94 kb fragment encoding methotrexate resistance from - -the plasmid pSV2dhfr (Subramani et al (1981) Mol.
Cell. Biol. _1, 854-864) and a 1.15 kb fragment encoding 6418 resistance from the plasmid pMClneo polyA -(Thomas and Capecchi (1987) Cell 51, 503-512).
The neo gene was obtained as an XhoI-SalI fragment and the dhfr gene was obtained by PCR amplification using the primers 5'-GGACGCGTGGATCCAGACATGATAAGATA-3' and 5'-GGACGCGTCAGCTGTGGAATGTGTGTCAG-3' designed to add MluI
sites at the ends of the resultant fragment. The neo and dhfr genes were cloned into the XhoI and MluT
sites respectively of pCG.CMV/EPO to give the plasmids pCG.CMV/EPO/DHFR and pCG.CMV/EPO/Neo/DHFR such that their transcription is in the same orientation as that of CMV. Finally, the 5 kb Ba~FiI-HindIII fragment from pTD.l was added via ClaI adapters at the ClaI site of pCG.CMV/EPO/Neo/DHFR to give pCG.HRl. In pCG.HRl, the 5' Skb EPO fragment is in the same orientation as that of the 620 by BstEII-XbaI fragment with respect to the original lambda clone.
A 9.54 kb fragment containing the 5' Skb BamHI-HindIII EPO fragment, the dhfr and 6418 markers, the CMV enhancer/gromoter and the 620 by BstEII-Xbal EPO fragment can be released from pCG.HRl as a NotI or SacII fragment. This NotI fragment can be used for homologous recombination as it is designed to serve as an omega structure in recombination having 5 kb and 620 by of homology to facilitate the event (FIG. 3).
For electroporation, the DNA was first cut with NotI, then extracted with phenol/chloroform and precipitated by the addition of ethanol before centrifugation. The resultant DNA pellet was resuspended at a concentration of 2 mg/ml in a volume (10 ~tl) of 10 mM Tris-HC1, 1 mM EDTA (TE).

Introduction of DNA into cells Transformed primary human 293 embryonal kidney cells {ATCC CRL 1573) were cultured in Cellgro*
DMEM H16 (Mediatech) supplemented with 10~ calf serum, glutamine (2 mM) and penicillin (100 U/ml)/streptomycin (0.1 mg/ml) and grown at 37°C
in 5$ C02. At 90$ confluency, cells were prepared for electroporation by trypsinization, concentration by brief centrifugation and resuspension in PHS at 107 cells/0.8 ml. The cells were equilibrated at 4°C, and DNA (50 fig) restricted with NotI (as described above) was added. The mixture was electroporated at 960 ~CF
and 260 v with a BioRad Gene Pulser and then iced again for 10 min before plating onto a 10 cm dish.
After incubation at 37°C for 48 hr, the cells from a 10 cm dish were split equally among 5 24-well plates in media containig 6418 at 0.6 mg/ml (effective concentration). Under these electroporation conditions, 4-10 colonies/well survive drug selection after 2 weeks.
Detection of HomoloQOUS Recombination by PCR Analysis Using NotI restricted DNA from pCG.HRl, successful homologous recombination is obtained by insertion of the 3.8 kb construct at the targeted EPO
locus while simultaneously deleting 1.2 kb of genomic sequence (FIG. 3). PCR is used to detect unique targeting events versus random integration of the DNA
as diagrammed in FIG. 4. Two primers are synthesized, one to the 3' end of CMV and the other to the region 3' to the XbaI site used for the 620 by BstEII-XbaI
fragment in the targeting DNA. A homologous recombination event generates a DNA target in the genome from which these primers produce an amplification product of 860 bp.
In order to detect the targeting event, pools of clones (from the electroporated 293 cells) *Trademark from 4 wells each (representing about 16 colonies) were generated by trypsinizing wells and using 90% of each well for the pool. The remaining 10% of each well was then reseeded back into the well. Genomic DNA was then prepared from each pool as follows. The cells in each pool were pelleted by centrifugation for 2 min. in a 1.5 ml microcentrifuge tube, resuspended in PBS (20 ~C1), and treated for 1 hr at 37°C with a solution (400 ~C1) containing 10 mM Tris-HC1 (pH7.5), 100 mM NaCl, 5 mM EDTA, 1% SDS and RNase A (40 ~Cg/ml).
Proteinase K (10 ~1, 10 mg/ml) was then added, and the samples were incubated for 4 hr at 50°C before extractions by vigorous vortexing with phenol/chloroform (200 ~C1 each), then with chloroform ( 400 ~C1 ) , the addition of ethanol ( 800 ~Cl ) , and centrifugation at 25°C for 10 min. The DNA pellets were washed with 70% ethanol, dried and resuspended in TE (20 ~1). An average of 40 ~Cg of genomic DNA was obtained from each sample.
Approximately 1 ~Cg from each sample of genomic DNA was used for PCR analysis. The DNA in a volume (10 ~1) of TE was boiled for 10 min. prior to the addition of PCR mix (40 ~1). The reaction (50 ~1) contained 10 mM Tris-HC1 (pH 9.0 at 25°C), 50 mM KC1, 1.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100; 200 ~M
dNTPs, 1 ~M each of the primers 5'-AAGCAGAGCTCGTTTAGTGAACCG-3' and 5'-TGAGCGTGAGTTCTGTGGAATGTG-3', and 1.5 U of Tag DNA
polymerase (Promega). Following an initial incubation of 94°C for 3 min, the samples were subjected to 45 cycles of denaturation at 94°C for 1 min., annealing at 66°C for 1.5 min. and extension at 72°C for 2 min.
At the end of the 45 cycles, the samples were incubated an additional 5 min. at 72°C. A portion (20 ~C1) of each sample was analyzed on a 1% agarose gel run in TBE and stained with ethidium bromide. Out of the 90 pools analyzed from 3 electroporations, two samples were identified which exhibited the correct *Trademark size fragment by ethidium bromide staining. The DNA
from the PCR reaction was recovered and subjected to restriction mapping with Xbal. The correct amplification, product should upon treatment with XbaI
yield two fragments, 669bp and 191bp. The samples from the two pools both yield fragments of the correct sizes. In addition, the sample from pool 1 exhibits other bands i.n the uncut material.
Fol.lowi.ng the procedure described previously, metaphase chromosomes are prepared from the recipients demonstrating homologous recombination with DHFR and transformed in DHFR deficient CHO cells.
After isolating resistant colonies and analyzing for expression o1: EPO, the cell clones are grown in selective medium containing progressively higher concentrations of methotrexate (.02-80 ACM) with steps of 4-fold increases in concentration. The cells are then harvestE:d, cloned and screened for production of EPO. Clones providing for at least 2-fold enhancement of EPO production are isolated.
It is evident from the above results, that the subject rnethad provides for a novel approach to expression o:E a wide variety of mammalian genes of interest. The method is simple, only requires the knowledge of a sequence of about 300 by or more in the region of a target gene, and one may then use substantiall~t conventional techniques for transferring the amplifiable region to an expression host, and production o:E the desired product in high yield.
35 Although the foregoing invention has been described in some detail by way of illustration and example for :purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in w~ ~no~~ Pcriusgoioba~
;,..
U .::
the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for producing mammalian proteins comprising:
transforming mammalian host cells comprising an endogenous target gene with a construct comprising an amplifiable gene and/or a heterologous nucleotide regulatory sequence and at least one flanking region homologous to a region of the host cell genome within or proximal to said endogenous target gene, so that the amplifiable gene and/or heterologous regulatory sequence are integrated via homologous recombination into the genome of the mammalian cells and the amplifiable gene and/or heterologous regulatory sequence become operatively associated with said endogenous target gene so that said endogenous target gene is amplified and/or so that expression of said endogenous target gene is controlled by said nucleotide regulatory sequence;
selecting for cells comprising said construct by means of said amplifiable gene or other marker present in said construct; and culturing said cells comprising said construct under conditions wherein the targeted gene is expressed and the protein encoded by the targeted gene is produced.

2. A method of integrating an amplifiable gene and/or a heterologous nucleotide regulatory sequence into the genome of a mammalian cell that contains an endogenous target gene comprising inserting said amplifiable gene and/or said heterologous nucleotide regulatory sequence, by targeted homologous recombination, into the genome of said mammalian cell wherein said endogenous target gene is amplified when said amplifiable gene is amplified and/or wherein expression of said endogenous target gene is controlled by said heterologous nucleotide regulatory sequence.

3. A method for amplifying gene expression in a mammalian host cell having an endogenous target gene comprising:

(a) transforming a mammalian host cell with an amplifiable gene, flanked by a nucleotide sequence homologous to a region of the host cell genome within or proximal to the endogenous target gene, so that the amplifiable gene is integrated by homologous recombination into the genome of the mammalian host cell; and (b) selecting a transformed mammalian host cell in which the integrated amplifiable gene is operatively associated with the endogenous target gene, so that the endogenous target gene coding sequence is not disrupted and so that the endogenous target gene is amplified and expressed when the mammalian cell is cultured under conditions that amplify the amplifiable gene.

4. The method of claim 3 further comprising inserting with said amplifiable gene a nucleotide regulatory sequence heterologous to said predetermined endogenous target gene, by targeted homologous recombination, into the genome of said mammalian cell so that expression of said predetermined endogenous target gene is controlled by said nucleotide regulatory sequence.

5. A method according to any one of claims 1,2, or 4 in which the heterologous regulatory sequence is a cytomegalovirus promoter/enhancer.

6. A method according to any one of claims 1,2,4 or 5 in which the endogenous target gene is normally not expressed by the mammalian cell.

7. A method according to any one of the claims 1 to 6 wherein the mammalian host cell is a primary cell.

8. A method according to claim 7 in which the primary cell is a fibroblast, lymphocyte, epithelial or endothelial cell.

9. A method according to any one of claims 1 to 8 wherein the target gene is a human gene.

10. A method according to claim 9 in which the target gene encodes an interleukin, a growth factor, a colony stimulating factor, erythropoietin, a plasminogen activator, an enzyme, an interferon or a receptor protein.

11. A method according to any one of claims 1 to 10 in which the amplifiable gene is dihydrofolate reductase, metallothionein-I, metallothionein-II, adenosine deaminase, or ornithine decarboxylase.

12. A mammalian host cell having an endogenous target gene comprising an amplifiable gene and/or heterologous nucleotide regulatory sequence, integrated by homologous recombination, into the genome of the mammalian host cell in operative association with the endogenous target gene so that said endogenous target gene is amplified when said amplifiable gene is amplified and/or so that expression of said endogenous target gene is controlled by said heterologous nucleotide regulatory sequence wherein said amplifiable gene is at other than its wild type site.

13. A mammalian host cell according to claim 12, being a human 293 embryonal kidney cell wherein the genome of the cell has inserted therein an enhancer and promoter of cytomegalovirus operatively associated with the human erythropoietin gene, so that the cell expresses human erythropoietin.

14. A host cell according to claim 12 wherein the target gene is a human gene.

15. A method for large scale production of a mammalian target gene product in cell culture, comprising:
(1) culturing a mammalian continuous cell line which was prepared by the steps of:
(a) integrating, via targeted homologous recombination, a nucleotide regulatory sequence heterologous to the target gene, into the genome of a mammalian host cell, so that the integrated nucleotide regulatory sequence is operably associated with the mammalian target gene contained in the host cell genome to form a recombined mammalian target gene; and (b) transferring the recombined mammalian target gene to a mammalian continuous cell line compatible with the integrated nucleotide regulatory sequence, so that the mammalian target gene product is expressed by the mammalian continuous cell line in culture; and (2) recovering the mammalian target gene product from the cell culture.

16. The method of claim 15 in which the mammalian continuous cell line further contains an amplifiable gene operatively associated with the mammalian target gene controlled by the heterologous nucleotide regulatory sequence, and the mammalian continuous cell line is cultured under conditions that amplify the amplifiable gene and the mammalian target gene, so that expression of the mammalian target gene controlled by the heterologous nucleotide regulatory sequence is enhanced, in which the mammalian continuous cell line was prepared by:
(a) integrating, via targeted homologous recombination, the amplifiable gene within an intron or proximal to the mammalian target gene contained in the mammalian host cell, so that the amplifiable gene and the heterologous regulatory sequence are operatively associated with the mammalian target gene; and (b) transferring the recombined mammalian target gene to a mammalian continuous cell line, so that the expressed mammalian target gene controlled by the heterologous regulatory sequence is amplified when the mammalian continuous cell line is cultured under conditions that amplify the amplifiable gene.

17. A method for producing a mammalian continuous cell line used for large-scale protein production in culture, comprising:
(a) integrating, via targeted homologous recombination, a nucleotide regulatory sequence heterologous to a mammalian target gene contained in a mammalian host cell, so that the integrated nucleotide regulatory sequence is operably associated with the mammalian target gene to form a recombined mammalian target gene; and (b) transferring the recombined mammalian target gene to a mammalian continuous cell line compatible with the integrated nucleotide regulatory sequence, so that the mammalian target gene product is expressed by the mammalian continuous cell line in culture.

18. The method for producing the mammalian continuous cell line of claim 17, which further comprises:
(a) integrating, via targeted homologous recombination, an amplifiable gene within an intron or proximal to the mammalian target gene contained in the mammalian host cell, so that the amplifiable gene and the heterologous regulatory sequence are operably associated with the mammalian target gene; and (b) transferring the recombined mammalian target gene to a mammalian continuous cell line, so that the expressed mammalian target gene controlled by the heterologous regulatory sequence is amplified when the mammalian continuous cell line is cultured under conditions that amplify the amplifiable gene.

19. The method of claim 16 or 18 in which the amplifiable gene is dihydrofolate reductase, metallothionein-I, metallothionein-II, adenosine deaminase, ornithine decarboxylase, or glutamine synthetase.

20. The method of claim 15, 16, 17 or 18 in which the mammalian host cell is a human cell.

21. The method of claim 15, 16, 17 or 18 in which the mammalian host cell is a primary cell.

22. The method of claim 21 in which the primary mammalian cell is a fibroblast, lymphocyte, epithelial or endothelial cell.

23. The method of claim 15, 16, 17 or 18 in which the mammalian target gene is a human gene.

24. The method of claim 23 in which the target gene encodes an interleukin, a growth factor, a colony stimulating factor, erythropoietin, a plasminogen activator, an enzyme, an interferon, or a receptor protein.

25. The method of claim 15, 16, 17 or 18 in which the heterologous regulatory sequence is a viral promoter or a promoter/enhancer.

26. The method of claim 25 in which the promoter/enhancer is a cytomegalovirus promoter/enhancer.

27. The method of claim 15, 16, 17 or 18 in which the mammalian continuous cell line is a Chinese hamster ovary cell line, a monkey kidney cell line, a mouse fibroblast cell line, a 3T3 mouse cell line, a VERO cell line or a 293 cell line.

28. A mammalian continuous cell line, the genome of which contains a heterologous genomic DNA encoding a gene product of interest operatively associated with:

(a) a nucleotide regulatory sequence different from the wild-type regulatory sequence normally associated with the heterologous genomic DNA; and (b) an amplifiable gene, so that expression of the heterologous genomic DNA is controlled by the regulatory sequence and is amplified when the mammalian continuous cell line is cultured under conditions that amplify the amplifiable gene.

29. The mammalian continuous cell line of claim 28 in which the heterologous genomic DNA is a human gene.

30. The mammalian continuous cell line of claim 29 in which the human gene encodes an interleukin, a growth factor, a colony stimulating factor, erythropoietin, a plasminogen activator, an enzyme, an interferon or a receptor protein.

31. The mammalian continuous cell line of claim 28 in which the regulatory sequence is a viral promoter or promoter/enhancer.

32. The mammalian continuous cell line of claim 31 in which the promoter/enhancer is a cytomegalovirus promoter/enhancer.

33. The mammalian continuous cell line of claim 28 in which the amplifiable gene is dihydrofolate reductase, metallothionein-I, metallothionein-II, adenosine deaminase, ornithine decarboxylase, or glutamine synthetase.

34. The mammalian continuous cell line of claim 28 which is a Chinese hamster ovary cell line, a monkey kidney cell line, a C127 mouse fibroblast cell line, a 3T3 mouse cell line, a VERO cell line or a 293 cell line.