JPS6112288A - Vector containing auxiliary dna for characteristic conversion of eykaryotic cell - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to the production of proteins of interest by eukaryotic cells. In particular, it relates to obtaining high yields of commercially useful proteins secreted from eukaryotic cell cultures transformed with genes encoding proteins and selectable markers, and more particularly to enhancing the expression of proteins. auxiliary DNA (acce
The present invention relates to a transformation vector containing a gene that coats a protein in association with ssory DNA).

The following definitions are provided to facilitate understanding of the invention. The following definitions should extend to culmness, which deviates slightly from the meaning prevailing in the art.

Amplification refers to the process by which cells cause gene replication within their chromosomal DNA.

Cotransformation refers to the process of transforming a cell with one or more exogenous genes that are foreign to the cell, one of which confers a selectable phenotype on the cell.

Downstream refers to the direction towards the end of the cleotide sequence.

An enhancer is a nucleotide sequence that is capable of enhancing the transcription of a gene, regardless of the nature of the gene, the position of the nucleotide sequence relative to the gene, or the orientation of the sequence.

A gene is a deoxyribonucleotide sequence that coats the mature protein of interest. As used herein, a gene will not include untranslated flanking regions such as RNA transcription initiation signals, polyazonylation sites, promoters, or promoters.

A selection gene is a gene that confers a phenotype on cells that express the gene as a detectable protein.

A selection agent is a condition or substance that allows the expression of a selected gene to be detected.

Phenotype refers to observable properties of a cell that are expressed by the cell's genotype.

A product gene (p) is a gene that encodes a protein product with desirable properties such as those useful diagnostically or therapeutically.

Genotype (g□>notype) refers to genetic information contained within a cell as opposed to its expression observed as a phenotype.

Bonding (1iga, tion) refers to the process of forming a phosphodiester bond between the 5' and 3' ends of two DNA strands. This is accomplished by several well-known enzymatic manipulations, including blunt-end ligation with T4 DNA ligase.

Orientation refers to the ordering of nucleotides in a DNA sequence.The opposite orientation of an eDNA sequence refers to the S/'i order of the nucleotides in a DNA sequence.
-37 order is one in which the sequence is reversed when compared to the norm of the DNA from which it was obtained. Reference points of this type include the direction of transcription of other specific DNA sequences in a DNA source or the origin of replication of a replicable vector containing that sequence.

Transcription refers to the synthesis of RNA from a DNA template.

Transformation means changing the genotype of a cell by incorporating foreign DNA into the cell. Transformation may be transient or stable, and the transformed cells, which in some cases can be detected by a change in the cell's phenotype, are called transformants. The pre-transformed cell is called the mother cell.

Translation (tr, anslation) by Metsenscher R
Refers to the synthesis of NA or lanopolypeptide.

Introduction of DNA into eukaryotic cells is generally well known and can be accomplished by a variety of standard methods. These include protoplast fusion, DNA microinjection, chromosomal transfection, lytic and non-lytic viral vectors [e.g. Mulligan et al.
e” (London) and E: 1O8-114 (1979)
), cell-cell fusion (Fournier et al. - Proc
, Nat. Acad. Sci,” 74: 319-3.
23 (1977)), lipid structure (U.S. Pat. No. 43944)
No. 48) and cellular endocytosis of DNA precipitates (Bachetti et al. - Proc, Nat, Aca
d, Sci.” 74:1590-1594 (197
7))).

Can cells temporarily uptake IIJA%?

Alternatively, the DNA can be incorporated to produce stable cell lines depending on conditions. One way to obtain stable cell lines is to obtain DNA containing the protein gene of interest.
Inserting a selection gene with A and keeping the cell lineage under selection pressure. In any event, it is desirable to find ways to enhance protein expression from transformed cells.

SUMMARY OF THE INVENTION The present invention provides a means to enhance the expression of heterologous proteins within eukaryotic cells. According to the present invention, a method for enhancing the expression of a heterologous protein in a eukaryotic cell includes introducing into the eukaryotic cell a DNA encoding a heterologous non-viral protein and an auxiliary DNA, and culturing the cell. It consists of expressing proteins.

The present invention provides transformation vectors containing auxiliary DNA for use in eukaryotic cells to express a single heterologous non-viral protein.

In a preferred embodiment, a gene expressing an amplifiable selectable phenotype is introduced into a eukaryotic cell to facilitate amplification of introduced DNA and auxiliary DNA encoding a heterologous protein of interest.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, auxiliary DNA and DNA encoding a protein of interest (ie, the product gene) are introduced into eukaryotic cells. Auxiliary DNA enhances expression of the intended protein. For example, the auxiliary DNA can increase the efficiency of transcription or translation, or have other effects to enhance expression of the protein of interest.

According to the invention, the auxiliary DNA is part of the transformation vector system. It is D that encodes the foreign protein of interest.
It is introduced into the cell before, simultaneously with, or after the introduction of NA. Auxiliary DNA is DNA that improves the stability of products synthesized by a transformed cell line, increases the amplification of foreign genes introduced into that cell line, and increases the efficiency of transcription or translation. The auxiliary DNA contains functions recognized by the mother cell. Thus, the auxiliary DNA can be more than just carrier DNA or bulky DNA previously used in transformation.

One class of auxiliary DNs, Ik, is the translation activator!
Consists of DNA that supports Translation activating genes produce proteins or short untranslated RNA products that interact with messenger RNA to increase the efficiency of translation.
Do t. One example is Virus Associated (MA)RN
Adenovirus DNA encoding A cTh
immap paya et al.'s "Ce1l" 31:543~
551 (1981)). This DNA is a small non-translated RNA
A produces two substances (vAl and VAz).

The RNA product of VA DNA is linked to the adenovirus maj or late promoter in a currently uncertain manner.
latepromoter ) in conjunction with the tripartite leader sequence.

It is thought to enhance translation from mRNA containing its leader sequence. l RNA also increases the translatability of other early adenoviral mRNAs [SVensson and Akusjaru, “MO Engineering, Ce1l Bio,”
4 Near 36-742 (1984)]. vAl t TAHA VA2 DNA HA It may also be transfected without being linked to the product gene or selection gene.

VA RNA-enhanced translation is most dramatic when the late adenoviral mRNA tripartite leader structure is present in the mRNA.

Another class of auxiliary DNA includes eukaryotic genomic DNA from the mother cell. This DNA is the origin of replication or D
Although it is thought to contain sequences that promote the stability of NA, the mechanism by which this type of DNA promotes its beneficial effects is unknown. This DNA is the genomic DNA of the cell lineage.
It can be obtained by randomly cutting A or digesting it with an endonuclease. Genomic supplement D
The NA should consist of fragments approximately 50-5000 base pairs in size. These fragments are then ligated at any available restriction site upstream from the promoter (preferably upstream from any promoter) or downstream from the polyazonylation site of the transformation vector. These vectors are thus suitable for transformation, after which the transformants exhibit one or more undesirable properties (e.g. stability of product synthesis after stepwise cultivation of the transformants and/or
or high yield of produce).

The yield of product from transformants can be improved by transforming the cells with a class of supplementary I) NAs termed trans-active transcriptional activators.

The species DNA encodes a protein or protein derivative that stimulates transcription. One class of transcriptional activators is termed immortality genes because they enable infinite successive progeny formation; cells containing this type of gene do not die after a predetermined number of divisions. These activators do not need to be bound within the same DNA strand as the product gene to increase the rate of transcription, and in this they differ from facilitators [Imperiale et al. Ce11"35: 127
~l 36 (1983), Green et al.
"35: l37-14s (1983):].

Several trans-component transcriptional activators for use in the present invention are themselves known and have been cloned. The most widely studied examples include human) C-myc, SV40 canine T antigen, polyoma large T, and adenovirus E.
Contains the IA gene.

The product genes that can be used are essentially unlimited. Genes that coat proteins or genes that encode substances that can be produced by protein-based reactions such as enzymatic conversion are suitable. Genes encoding proteins (e.g. some enzymes from prokaryotic or lower eukaryotic sources) that adversely affect the host cell culture by synthesizing or hydrolyzing host proteins can be used, e.g. It can be used with restrictions, such as adding to or selecting a lower expression level than is considered optimal. Genes for active proteins or enzymes are found in the cells of mammals or higher animals such as vertebrates. Most interesting genes encoding therapeutic proteins would fall into this category.

Examples of product genes include blood coagulant or fibrinolytic proteins (e.g. antihemophilic factor, tissue plasminogen activator, urokinase and coagulation factors ■, ■, lX, X
or Cell active substances such as lymphocytes, growth hormone and platelet-inducing growth factors), oncogene products, cell surface antigens, immune proteins (
eg gG, gE and gM) and complement, as well as other genes encoding proteins of commercial interest.

Vector for transformation 1 The vector used for transformation according to the present invention is generally auxiliary D.
It will contain the NA and the product gene. Furthermore, the × lid for transformation usually includes a promoter, an intron + a horiadenylation site, and a 3'
There may be other elements such as uncoated 9 areas. If a selection gene is used, it can be linked to the product gene in the vector, or it can be present in a separate vector that is co-transformed with the lid containing the product gene.

Selection genes are divided into three categories: detectably amplified selection genes, dominant selection genes, and detectably amplified dominant selection genes.

A selection gene that is detectably amplified is one whose amplification can be detected by exposing the host cell to a selection agent. A non-dominant gene that is detectably amplified generally requires a maternal line lacking the selected gene as a genotype. Examples include asparagine synthetase; aspartate transcarbamylase [Kemp et al.'s “Ce11”9:
541 (1976)); azo=7t, acid deaminase [DeBatisse et al. -MolandCell B
iol,” 2(11): 1346-1353(19
82)); adenosine deaminase (J of Yeung et al.
, B, C. 258: 15185 (1983)];
Includes genes such as mouse dihuman 90 folate reductase (DHFR) and mouse thymidine kinase (TK) (with a deleted promoter).

A dominant selection gene is one that is expressed in the transformant regardless of the genotype of the mother cell.

Most dominantly selected genes are not detectably amplified because their phenotype is so effective at processing the selection factor that cell lineages that amplified the gene and cell lineages that did not amplify it. This is because what is being identified is a cone. An example of this type of dominant selection gene is xanthine-guanine phosphoriboxyltransferase [Mulli
'Proc of gan et al.

Nat, Acad, Sci,” 78(b)): 2
072-2076 (1981)) and aminoglycoside 3'-phosphotransferase [Colbere
-J0Mc+1 of Garapin et al. B101. ”1
50: Shi 14 (1981): ], these are genes that coat enzymes in prokaryotes.

Some dominant selection genes are also detectably amplified. A suitable example is Haber et al.'s “Somat, 1cCp
HGenet'±: 499-508 (1982)
the mutant DHFR gene described in 1999, cell surface markers such as HLA antigens, and enzymes (e.g., specific esterases) that produce fluorescent or colored products from fluorogenic or chromogenic substrates known in the art. Contains genes encoding.

Preferably, a dominant selection gene that is detectably amplified is used in the present invention. In some cases, a dominant selection gene is determined by appropriate mutations within that gene.
It should be understood that it can be converted into a gene that is detectably amplified.

Preferably, the selection factor is one that inhibits cell proliferation in the absence of the selection gene. Thus, revertant cells in long-term culture that lack the selection gene (and possibly the product gene) or no longer express the selection gene will not overgrow their population. However, in the commercial production of protein products for therapeutic purposes, it would be desirable to avoid the use of cytotoxins and simplify the purification process of the product. Therefore, a desirable selection gene would be one that would allow the transformant to use nutrients essential for growth that it would not be able to access if it lacked the selection gene. One example is the TK gene mentioned above.

It should be noted that the selection gene can also be a product gene. For example, it is desirable to harvest the product of a selected gene for use as a therapeutic or diagnostic agent. A product gene will become capable of functioning as a selection gene if the environment of the transformant is altered so that the product can confer some selection benefit to the transformant. . For example, a product gene can produce an enzyme that acts on an otherwise unavailable essential substrate in the cell culture medium to release the essential substrate (eg, an essential nutrient).

The selection genes and product genes used herein may carry all or a portion of their wild-type untranslated flanking sequences, but normally such sequences will not be carried as described below. In the case of linked vectors, particularly beneficial results in product yield are obtained by locating the product gene upstream from and in close proximity to the region of translation of the selected gene. This generally indicates that most, if not all, of the 3' untranslated region found downstream from the wild-type product gene and the 5' untranslated region found upstream from the wild-type selection gene are cleaved by the appropriate restriction endo9 nuclease. or cleaved by oligonucleotide priming-mediated deletion at M13 as described elsewhere herein. Result 1
Both genes are directly linked with no promoter inserted between the product gene and the selection gene.

Genes used herein will generally include a wild-type translated sequence encoding a prepropolypeptide (eg, a secretion leader) that is desirable for the structure, stability, and/or secretion of the mature product. However, if the wild-type translation leader sequence does not function properly in the transformant, this type of prepro sequence may be deleted prior to integration into the vector and subsequent co-transformation of host cells, or It may be substituted by other prepro sequences that work properly in the jar.

In general, the interruption of a vector or a gene contained within a vector with an intron means that the Metsenger RNA transcript from the vector is properly excised and translated into the mature protein and desired leader sequence by the transformant. Sanger RNA is not important as long as it occurs. This would be the case for introns found in a given higher eukaryotic gene that would be processed by other higher eukaryotic cells. Since the gene is a CDNA reverse transcription product, there are no introns at all, and the post-
tra, n5script, 1onal splicin
The probability of g) will be considerably reduced.

In the case of linked vectors, the coding strands for the selection gene and the product gene are preferably joined by directly joining the stop codon of the product gene to the start codon of the selection gene. Apart from this, both of these genes are linked via an oligonucleotide bridge rich in guanine deoxyribonucleotides (G) and cytosine deoxyribonucleotides (c). This bridge is R
To reduce the possibility of NA hairpin loop formation, a stop cobin and a disclosure codon, as well as a palindrome'! It is better not to include l'.

The terms "product gene" and "selection gene"
This does not imply that only one each gene or only one each gene code is used within the vector. First, a given selected phenotype may require the synthesis of one or more distinct proteins. In this case 1, for example, the selection gene for each protein is present in a vector that is not covalently linked to the product gene or to any other selection gene, and each selection gene is as described above or in U.S. Pat. It will be linked to the product gene as described in No. It is undesirable to use more than one selection gene if each selection gene confers the selection phenotype for the same selection factor independently of any other selection gene.

It may be desirable to transform using one or more jars containing multiple distinct product genes. Of particular interest are distinct product genes that encode proteins with mutually beneficial effects. For example, proteins that stabilize other protein products, or that are part of a multi-protein system that has a biologically active effect, can be co-expressed.

Additionally, either or both of the selection gene and the product gene may be repeated within one or more vectors, ie, present in multiple, generally tandem, copies.

In such cases, it is advantageous for the repeated genes to each contain all of the RNA processing and transcriptional control sequences present in a vector containing only one copy of the gene.

Vectors can also include facilitating factors.

Facilitators are functionally distinct from promoters, but appear to operate in concert with promoters. Although their function at the cellular level is not well understood, their unique property lies in their ability to activate and enhance transcription regardless of location or orientation. The promoter must be present upstream of the gene, whereas the promoter must be present upstream from the promoter, i.e. in the 5' direction, within the gene as an intron, or downstream from the gene, i.e. the polyazonylation site, between the gene and the polyazonylation site. It may exist in the 3' direction. The reversal promoter is not functional, but the reversal promoting factor is. Promoting factors are /su-agonistic, ie, they have an effect on the promoter only when they are present on the same DNA molecule. For a general overview of promoting factors, see Kl)oury et al.'s “Ce
11” 33:313-314 (1983).

Suitable promoters are obtained from animal viruses such as simian virus 40, polyoma virus, bullion papilloma virus, retroviruses or adenoviruses. Ideally, the promoting factor would be from a virus that is tolerated by the host cell, ie, a virus that normally infects cells of the host type. Viral promoters can be easily obtained from publicly available viruses. For example, the promoter regions of several viruses, such as Rous sarcoma virus and simian virus 40, are well known.

Lll ci ev et al.'s “Ce1l' 33: 7
05-716 (1983) C. It is conventional to excise these regions based on the published restriction map of the virus in question, modify those sites as necessary, and tether the promoter into a vector for the product or selection gene. Probably chemical means◇
For example, Mo1 of Kaufrnan et al. Ce1l B
iol, 21304-1319 (1982). Alternatively, promoting factors can be synthesized from sequence data. The size of viral promoters (generally about 150 base pairs or less) is small enough that this may be practically achievable.

Facilitators can be repeated within the vector either in tandem (when using the naturally occurring SV40 viral facilitator) or segregated throughout the vector at the sites discussed above. Preferably, the promoting factor has the same directionality with respect to the product gene and/or selection gene as it had with respect to the gene under its influence in the wild-type source. Preferably, the promoter is located upstream from any promoters present within the vector. Multiple different promoting factors may be used.

Also, promoting factors do not need to be linked to a product or selection gene to be useful during transformation. In unlinked vector systems, the promoting factor is preferably present in the vector containing the product gene. This will increase the likelihood that the product gene and selection gene will be physically linked within the co-transformant. In this type, about 1 to 100 times more transformants can be obtained with a predetermined amount of transforming DNA, depending on the promoter used for transcription of the selected gene.

The promoter need not contain any of the flanking sequences found in its wild-type environment, such as the viral origin of replication, relevant promoter components such as the TATA box, cap sites or transcription primer sequences. However, it may be advantageous to incorporate all or part of these sequences when restriction enzyme sites do not exist to allow deletion of these sequences. It may also be more convenient to use a DNA fragment that contains both a promoter and a promoter under wild-type control of the promoter.

Facilitators and facilitator-promoter regions can be selected from eukaryotic cells rather than viruses. Preferably, these are promoting factors associated with genes that produce large amounts of protein in the source cell. They were found to similarly produce high yields of products from genes other than the direct genes normally regulated in the wild type environment. The host cell to be transformed is preferably of the same somatic or germline lineage as the cell from which the promoting factor was obtained. For example 1. A region that activates or promotes immunoglobulin genes within the Tk-Ck intron (immunoglobulin promoter) is introduced upstream or downstream of the product gene, and the construct of and 5 is introduced into the myeloma cell lineage along with the selected gene. be introduced. For details about this promoting factor, please refer to G11.
"C+all" and near 17-728 (19
83). Is immunoglobulin promoting factor a protein product? Advantageously, it is used in vectors that are intended to be inserted into myeloma cells for production.

Both the product gene and the selection gene will be linked to the promoter so that they are under the transcriptional control of the promoter (except in the case of linked vectors as described above).

The promoter may be the wild-type promoter for the gene in question, or the gene may be linked to a promoter from another gene, a progeny, in another eukaryotic cell lineage or a cell lineage transformed from a prokaryotic virus. Good too. Obviously, the promoter should be recognized by the host to be transformed without accidental recombination with the promoter in the transformed cell, but otherwise the choice of promoter is not considered limiting. Particularly desirable promoters are those that are linked to a 5' untranslated leader and are transcriptionally activated by exogenous factors or conditions such as accessory gene products (transcripts and polypeptides), heavy metal ions, heat shock or viral infection. or translationally activated. A preferred promoter for product gene expression is the adenovirus major late promoter with a tripartite leader.

The vector system may be a promoter if a promoter unrelated to the promoter (e.g. the promoter for mouse alpha globin) is selected. There is no need to include it. However, rather than such promoter-independent strong promoters, vectors containing promoters and promoter-dependent strong promoters (eg, the adenovirus major late promoter) are used. A strong promoter is one that under controlled conditions produces the same or more transcripts than the SV40 early promoter.

Another element that may be desirable to be present within the vector is a polyabnylation site. This is a DNA sequence located downstream from the translation region of the gene, and an adenine ribonucleonucleotide is added to the gene to create the 3rd generation of Metsenger RNA.
Forms a polyadenylate tail at the ′ end. Polyazonyray-7 is important in stabilizing Messenger RNA against cellular degradation, a phenomenon that reduces Messenger RNA levels and protein product production.

Polyazonylation sites in eukaryotic cells are well known. A consensus (consensυS) sequence exists between eukaryotic genes: 57-
AAUAAA-3' is found 11-30 nucleotides from one point in the RNA from which the polyabnylation starts. DNA containing a polyadenylon site
Sequences can be obtained from the virus according to published reports. Examples of polyadenylon sequences are derived from the mouse turglobin and the late or early region genes of simian virus 40. Viral polyazonylation sites are preferred. Since these sequences are known, they can be synthesized in vitro and ligated into vectors using conventional methods.

The polyazonylon region should be located downstream from the product gene in a linked or unlinked vector. In an unlinked vector it is optionally linked downstream from the selected gene. The sequence separating the polyazonyl-/y/o/ site from the translation stop codon is preferably a region of untranslated DNA, such as a non-promoted eukaryotic gene. The oligonucleotide is terminated (from the E coton to the polyazonylene
Preferably, it extends a considerable distance (less than about 1000 bases) to the John site. This 3' untranslated nucleotide sequence generally increases product yield. The vector terminates approximately 30 base pairs downstream from the consensus sequence, but the gamma
It is advantageous to preserve the alignment.

These sequences generally extend about 200 to 600 base pairs downstream from the polyazonylation site.

It was found that the presence of an intron in the untranslated and transcribed portion of the lid increases the yield of the product.

Introns of this type may be obtained from host cells or other sources than genetic sources. For example, inserting a 3' splice site from an immunoglobulin gene into the untranslated region of the adenovirus major late transcript in place of a portion of the normal intron of the kidney transcript can result in increased yield of product.

Cells transfected with transcriptional activators, translational activators, and strong promoters or a combination of promoters and promoters have the most enhanced product synthesis? provide. Cascade 9 effects will result from this transcriptional and translational activator-transformed cell.

The vector will suitably be a supercoiled double-stranded circular structure. This is the form in which the vector is obtained from standard prokaryotic cloning techniques (in which the lid is created). However, the vector may also be linearized, ie, covalently cleaved at one point, concomitant with other steps such as ligation to genomic auxiliary DNA.

The following table is representative of suitable transformation vector systems.

Table 1 3. GF'-En-VP-Pa-p(A)-RG
F; GF'-8-p(A)-R-GF' 4, GF-EP-Pd-, p(A)-at;";l
"Go Shoulder Surface GF'-'ITA-In-VP-Pd-p
(A)-GF; order f Shimoishikan J rotation 11, EP-Pd-
B-8-E: n-p(A)”R; VP-TLk-p(
A)-R12, En-VP-Pd1-Pd2-3-p
(A)-R14,0F'-TLA-'VP-TI
'A -p(A)-R-GF -En -VP -Pa
-I -p(A>-R-GF 15-Aiko; VPI-Pd-p(A)-R; Koroshiko i-face possible 1 EP = non-viral promoter recognized by eukaryotic host cells VP- 712 viral promoter recognized by eukaryotic host cells 'ITA =) lance-acting transcriptional activator En-enhancing factor TLA = translational activator p(A)-#' reardenylation site pa = product gene S - selection gene B - oligonucleotide 9 bridges = phosphodiester bond; - unbound vector component?represents GF' - eukaryotic genomic fragment foreign intron R-4Jj component (plasmid or bacteriophage remainder from synthetic vector ) Shows the circular DNA sequence of a plasmid.

Vectors Linked vectors with short oligonucleotide 9 bridges or no oligonucleotide sequences between the synthetic product gene and the selection gene can be made in several ways. If there is no restriction site adjacent to or closest downstream from the translation stop codon of the product gene, the product gene is cleaved at the restriction site upstream of the wild-type translation stop codon; The codon is then linked to the selected gene fragment using a synthetic linking agent.

Another way to create such vectors would be to use bridging nucleotide probes to delete undesirable sequences between the product gene and the selection gene. Alternatively, if the carboxy-terminal amino acid of the protein product is not required for biological activity, a synthetic termination cognate may be attached to a convenient 3' end. The use of gene fragments here is not a preferred embodiment since it is desired to synthesize a protein product that is as identical as possible to that found in nature.

The 5' untranslated region that flanks the selection gene in the wild-type environment can be removed in the same manner as described above for the product gene, and this can be removed from the product gene as shown in Example 4. It occurs at the same time as the deletion. however,
The protein expressed by the selection gene or its derivative need not be identical to the wild type, for example, it may be more active against growth-limiting substrates or more resistant to cytotoxins than the wild type protein. It's fine. Preferably, the linkage is in the lid and the selection gene is under the control of the promoter of the product gene. This will help to stabilize the genotype of the cell and the lack of expression of the protein product, since cells lacking the product gene Y will not be able to survive the selection factor in the absence of a recombination event with the transformant.

The opposite was found in the case of unlinked vectors, but here too the same promoter can be used for both the selection gene and the product gene.

Alternatively, the vectors described herein can be synthesized by those skilled in the art using techniques known to those skilled in the art. Product genes, selection genes.

Vector components such as promoters, promoters, and auxiliary DNA (e.g., trans-acting transcription activators, translation activators, and the like) can be obtained from natural sources or synthesized as described above. You can also do that. Basically, vector components are DNA that can be obtained in large quantities.
If they are found within (e.g. components such as viral functions) and if they can be synthesized (e.g. polyazonyray-7 sites), then the vectors of concern will be able to culture the biological source and make the sonoDNA%'ia appropriate. Digest with endonuclease, isolate the DNA fragments, and extract DNA containing the element of interest.
The DNA can be obtained by identifying and recovering the DNA using methods well known in the art. Generally, transformation vectors are produced in small quantities and then ligated into autonomously replicating synthetic vectors, such as prokaryotic plasmids or phages. The pBR322 plasmid can be used in many cases. See Kaufman et al. (1982), supra. A large transformation vector is rosmid (cosmid: fur').
Although the DNA is encapsulated within a fascicaput, synthetic vector components may be required due to the carrying capacity, such as vectors that replicate as plasmids (vectors) when transfected into a recipient host.

Synthetic vectors can be prepared by conventional methods, such as transfection of recipient prokaryotes, synthesis to high copy numbers by replication of lids, recovery of synthetic vectors by cell lysis, and from cell debris by methods well known in the art. synthesis is by separating the lid.

Used to clone transformation vectors.

The resulting synthetic vector can be directly transfected into eukaryotic cells, or it can be isolated from the synthetic vector for transformation by digestion with an appropriate endonuclease, separation by molecular weight filtering, and collection of the lid for transformation. Isolation of the transformation vector is not necessary unless the remainder of the synthetic vector adversely affects the amplification, transcription, or translation of the eukaryotic gene. For example, a suitable synthetic vector for use herein is a plasmid). It is a mutant of psR322, in which sequences harmful to eukaryotic cells in plasmid pSR322 have been deleted.

Lusky et al. Nature 293: 79~
81 (1981). Use of this mutant obviated the need to delete the remainder of the 7 lasmid before transformation.

Transformation The cells to be transformed may be eukaryotic cells, including yeast cells, but are preferably cells other than fungal cells. Primitive transplants (including relatively undifferentiated cell lines such as stem cells) and immortal and/or transformed cell lines are suitable. Does a cell only have a selective gene that acts dominantly when it is used? There is no need to lose it as a genotype.

In some embodiments, stable transformed cell lines are not required. In such cases, suitable transformation vectors can be relied upon for the temporary introduction of DNA into mammalian cells (P, Mellon).

V, Parker, Y, G1+rzman
, T. Maniatis Ca1127: 2
79-288 (1981)). Isolation of a transformant of interest does not require that all cells of the population stably contain the exogenous gene expressing the protein product of interest. It is possible to introduce exogenous genes into subpopulations of cells that express the desired product over a period of several days. Since no selectable marker is required in the transformation vector by the DNA transfection and expression system according to the invention, the exogenous gene can be
Lost during cell proliferation over two weeks. but.

The synthesis of the desired product is found and detected 2-3 days after transfection of appropriate mammalian cells.

Thus, the host-vector system for cloning genes of mammalian proteins is the origin of replication-defective SV4.
Based on the development of a CV-1 monkey cell line transformed with Q DNA molecules (Y, Gluzman's Ce1
1.23:'175-182 (1981)). SV4
0 is a small DNA that replicates cytolytically in monkey cells.
It is a tumor virus. In the absence of DNA replication, SV
40 will transform monkey i1n cells. Defective Sv
Transformed monkey cy-1 cells (referred to as cOS) containing 40 DNA2 contain a complete copy of the SV40 genome [but produce high levels of large T antigen and S
V 40 Allows DNA modification. They also efficiently support replication of SV 40 with early region deletions and bacterial plasmids containing the SV 40 origin of replication [
PNA of R, M, Myers and R, Tjian.
S 77:649]-6495 (1980)). Therefore, this system allows the protein expressed by exogenous DNA ρ.
Exogenous DNA transfected via SV40-mediated DNA restoration to increase RNA rail υr1
A (Protein gene and auxiliary DNA) '& provides means for amplification.

In other embodiments, the cell is a stable immortalized mammalian cell line. Cell lines known to stably integrate the selection gene into their chromosomal DNA are suitable, such as the Chinese hamster ovary (cHO) cell line. In addition, there are He1a cells, CO8V cells, Me2/-Maaffg lineage (e.g., Boies M cell lineage), mouse L cells, mouse A fibroblasts, mouse N-type H3T3 cells, and similar cells. cells are useful.

Transformation with unlinked DNA can be performed stepwise or simultaneously. Methods are known to promote uptake of DNA by cells. Although microinjection of the vector into the cell nucleus will result in the highest transformation efficiency, it will generally be advantageous to expose the host cell to DNA in the form of a calcium phosphate precipitate. Product? The frequency of expressing transformants may be further increased by transforming with a molar excess (about 10:1 or more) of the product gene over the selection gene.

Selected transformants are then screened for binding of the product gene within their chromosomes or for expression of the product itself. The former is Jozan (Souther+)
This is accomplished using plot analysis, the latter using standard immunoassays or enzymatic assays.

Once the transformants are identified, a step is performed to further amplify the expression of the product gene by subcloning in the presence of constant or increasing amounts of the selection gene. Generally, this involves removing a population of transformants,
and (a) selecting one or more cells from the population that express the product in a superior manner when compared to other cells in the population; and (b) selecting the selected one or more cells. culturing the above cells into the next cell population under conditions set to select for changes in one phenotypic expression; and (c) step (b) 1: comparing to other cells of the population of J. et al. Step (b) involves the further selection of 1" or more cells from the population that express the product in this way; step (b) involves the selection of a large number of clones from step 8(a). This method is advantageously carried out by using the method described in pending U.S. patent application Ser.
is described in detail.

The following examples are provided to further illustrate the invention and are intended to be within the scope of the claims. It is not limited.

The temperatures used in the examples are degrees Celsius.

Example 1 Cell culture SV40 fl conversion C08vle cells (Rio-7M6)
is M+Horovitz (Horovitz et al., J.
Mo1-App-Genst, 2.147-9J-:)
(1983)). Adenovirus 2 was kindly provided by P.5harp. Adenovirus infection was carried out by absorbing 20 pfu/cell for 90 minutes at 37°C. After washing, cells were incubated at 37°C for 18 hours. A significant cytopathic effect was observed at this time.

DNA transfection was carried out by sompayrac and I) anna! 7Proc, Natl, A
cad, Scj,,,78.

7575-8-”-) (19'81) with chloroquine treatment (Luthman and Magn.
uSSOn.

Nuc, Ac1ds Res, 11. l 295-1
308 R-di (1983)). Cells were washed with serum-free medium and DEAE-1i'xtran (NW 5000.
000Pharmacia, 250 mg5) and 2 μq/ml plasmid)” DNA containing D
ul, becc.

Incubate in modified Eagle medium for 12 hours at 37 degrees. After incubation, cells are washed with serum-free medium and treated with 0.1 mM dichlorone in serum-containing medium for 2 hours at 37 degrees. Cells are then seeded in medium and incubated for the indicated time.

The plasmid constituent adenovirus-specific interacting factor is adenovirus M.
In order to analyze the n effect on LP, a series of DHF
The RcDNA Q gene is constructed, and they are shown in Figure t(a
) -t(e). pAdD 268V,A
(31 is the 7 first leader and adenovirus late m
Contains mouse DHFRCDMA Y under the control of adenovirus MLP containing the 5' splice site from RNA.

This 5' splice site is derived from the mouse immunodaloprine gene (Kau fman and 5harp', Mol
, Ce1l Biol,.

1304-19 (1982)).

The SV40 early polyadenylation signal is present at the 3' end of the DHFR coding region. cDNA genes contain sequences that are detrimental to the replication of mammalian cells (Lusky et al., Na, ture (L)).
ondon) ; 293, 79-81 <-') (
1981) "r-deficient, SV40-derived mata are clones (Mellon et al.) fX: pBR322 derivatives (p
Clone into SVOd, ).

Plasmi l' pAaD 263VpA (3) (Figure i
(a)) and pcVsVL (Fig. 1(b))
has already been described (Kaufman's 5 ha
r'p, supra). pCVSVL2 is identical to pCVSVL with the exception of the addition of an Xhol linker and the addition of a duplicate SV 40 SV40AvaI that was introduced by insertion into the upstream Xh○] site (15,83m, u, .

Is the SV40 late promoter in the same orientation as Aa 2 MLP? As shown, both AVa engineering ID fragments are also oriented.

pcVsVL2 hapAaD26sVpA(3) 7)
”) Kid4 Sah Luca L Kashi, 5V from psVod
40 products missing, 5V upstream of adenovirus MLP
40 Ennocer and origin of replication? include. pD2o
(Figure 1(c)) and pD17 (Figure 1(d))
is a CD for adenovirus fiber tannoprotein
Adenovirus sweat, 2nd and 3rd leader T codes derived from NA clone 138bp DNA
sequence (see Zain et al., 16,851-61'!
-) (1979)) pAd26s'' except for O insertion'
Jl) A(3) (Fig. 1 fa)). pD
20 is 8 bp 5' PvtxII at the 5' splice site
If the 138 bp region is inserted into the site in the correct orientation. Therefore, pD20 is the complete first . Leader of 2nd and P3 glue 3? encoded spliced late mRNA
A7: Contains 170 bp of Shiranaga J, and includes Sbp from the first late leader to the 5' splice site. pD17
(Figure t(a)) contains a 138 bp fragment in the opposite direction. pD61 ([kl(e)) is similar to pD20 (Figure t
(c) The third leader sequence of a2 present in )4
Identical to pcVsVL2 except for the inclusion of pcVsVL2.

pD17 and pD20. pJAW43 (Zain et al., supra)? To obtain D
NA polymerase 1 ty) Klenow fragment followed by digestion with PvuII. 138bp
Fragmen H isolated and previously digested with PvuII p
AaDzssVpA3). DNAQ Coliform H
BIOI and screened with the l38bp Xhol-PvtJI fragment from pJAW 43, which was radioactively recognized by eJ. Prepare DNA from positive hybridizing clones.

After digestion with PvuII, examine the orientation using acrylamide 9 gel electrophoresis. pD20 contains the 138 bp fragment in the same direction as the adenovirus MLP transcription direction, and pD17 contains the fragment 4 in the opposite direction. EcoR of pCVSvLρ・et al.
XhoL portion approximately 1, iKB fragment '7EcoR
pD61'4 is constructed by inserting the fragment from large pD20 completely digested with 1 and Xbol. The generated plasmid pD61 contains the SV40 engineering structure and a three-part leader. Adenovirus promoter with? contains.

These recombinants are used for research on adenovirus factors without affecting the expression of these cDNA genes. The experimental steps included DNA transfusion and adenovirus superinfection of COS monkey cells.

36 hours after DNA transfection, transfected cells? Infect with adenovirus and leave to incubate for 18 hours. Then the cell, a,
DHFR synthesis by labeling with 35B methionine for 1 hour, immunoprecipitation and polyacrylamide gel electrophoresis? The synthetic stone analysis result is AaD26SVpA (3) Oj O: pc
It was confirmed that DHFR synthesis was possible compared to the transfected sample (VsVL2). In mock-transfected CO8 Itoda cells, it is possible to detect chill DHFR, which has moved just above mouse DHF'H.

pcVsVL2 1st (j pcVsVL Kara (D
DHFR expression %'x: By transformation of CHODHFR cells, no differences were observed between the two plasmids when compared with cos cells. Furthermore, co-infection with adenovirus mopAdD26sVpA (3)'!
pCvSv showed almost no effect on DHFR synthesis.
L27! J) Lano expression is pAdD26sVpA (3)
(7) It was more than double. This is the result of the introduction of the upstream KSV40 enhancer of adenovirus MLP in pcVsVL2. A plasmid containing a 138 bp leader insert from a spliced adenovirus tripartite leader (pD20, p
D17 and pD6't) were also observed to synthesize DHFR.

Furthermore, both pD20 and pD61 respond to adenovirus infection with a 3-10 fold increase in DHFR synthesis in separate experiments. On the contrary, 13 in the opposite direction
pD containing 8 bp three-part leader segment
17 This expression is not affected by adenovirus co-infection. Could these results indicate that the sequences in the tripartite second and third leader segments respond to adenovirus infection in an orientation-dependent manner? Suggests.

To determine whether mRNA levels are affected by adenovirus infection, Kaufman et al.
1. cθ11 Biol, -King 1304-1319 (1
3'Sl mapping was carried out using the 3'-end labeled DNA probe as described in 982), and transfected and adenovirus-coinfected COS.
Total RNA isolated from monkey+jlH follicles and hybridized. F, >Sumi)'pCVSN'L2, pD
A single 550 base pair fragment was observed in pD61 and corresponds to the mRNA polyadenylated at the SV40 polyadenylation site. SV40 length? Missing < Is pD20 an sv4 Q enhancer? Approximately 2-fold lower DHFR% heterogeneous mRNA compared to the plasmid containing plasmids is shown. No change in the DHFR mRNA'A rail was observed from any cDNA gene even after adenovirus superinfection. This suggests that the increase in DHFR synthesis caused by adenovirus superinfection is due to DHFRmR.
This shows that this is not due to an increase in NA.

Increased DHPR synthesis upon adenovirus superinfection is a result of increased translation efficiency.

Example 2 To analyze the effect on MA RNA translation.

A new set of plasmids expressing human gamma interferon (Figures z(a)-2(d)) was constructed. Oligo dG-oligo dC tailing method (Mani
cDNA encoding human gamma-interf x C17y cloned from mRNA isolated from human peripheral blood lymphocytes by
S, Dr. C1ark (Genetic I'n5ti
Provided by tute)? I received it. The Pstl clone encoding gamma interferon was previously SV4.
The Pst site 3' of the 0 polyadenylation signal was eliminated. Insert into the P, tI site of the pcvSVL2 derivative.

Resulting plasmid p(gaff7-IF)D-5
(Figure 2 (a)) is the gamma-interface top coat 9.
3' of the 3' splice site of the 5' of the region DHFR cDNA and the rounding of the mRNA continuation? include. A similar plasmid with a three-part leader? To obtain both p(gamma-IF) D-6 and pD61, 1￥5all
and digested with Egl. 5all digest each plasmid once within the Tet2?icrin resistance gene of pBR322, and BglE digest each plasmid once just 5' of the 3' splice site from the immunoglobulin gene.p( Gamma-IF')
D-(, gamma F-containing fragment from 3
The partial leader containing fragment pD61 is ligated and the DNA is then used to transform E. coli HB1012 tetracycline resistant. pL58 (Figure 2 (b)
) contains the triad of azoviruses, but is otherwise identical to p(gamma-IF')D-5.

Adenovirus VA gene is Hind Engineering IIAd 2B
The fragment was isolated and introduced into pL58. Adenovirus HindAE IB fragment (17,1r
ru -31,7mu) was previously cloned into pBR,322 and binds to the Eco R function of pBR322 at the HindI site of sepJ, 31.7mu.

Digest this plasmid with °γHpa engineering (28,0 mu) and add the EcoR linker.

After EcoRI digestion, 1. LKB band? isolated, pL5
8 into the EcoR site. PQ2 (Figure 2
(c) In ), the VA gene is present in the vector, but
Transcribes in the opposite direction to adenovirus MLP. pQ3(
Figure 2(d)) is identical to pQ2 except that the VA gene is in the opposite orientation.

Gamma interferon The gamma-interfen assay was performed using CCU, 54 m cells (
(Stewart
, W.E., I., Interferon System.

Springer, Berlin, 1979). Thea'
? - Interferon units are expressed relative to the NTechH alpha interferon standard. Chloramphenicol acetyltransferase was described by Corman et al. in Mol, Ce1l Biol, 2, 1044.
-1051 (1982) from cell extracts.

The expression of gamma-interferon allows for a sensitive test of the quantitative effects of MA RNA. p-gamma
IF-5 and pL58 are identical to pD20 and pD61 except that the gamma-interferon c DNA has been inserted in the proper orientation of the ps tI function between the 3' splice site and the DHFR cDNA. Finally, pQ2 and pQ3 are located at the FCOR site of the adenovirus MA gene (adenovirus map unit) 28.
．． 02 and 31.00) are inserted in both directions.

Transformation in COS cells Plasmids pQ, 2, pQ3, pL58 and p-gamma er-6 are each transfected into CO8 cells (Table IA). After 36 hours of conditioning, a sample of the medium is assayed and one of two duplicate plates is infected with adenovirus. Tested 20 hours after infection, gamma-IF activity was detected again? Test. p-(gamma-IF')D-
5 showed low activity and was only slightly increased even with adenovirus coinfection. pL58 showed several times more activity than p6, and adenovirus infection showed a two-fold increase in gamma-IF activity. In contrast, both pQ2 and pQ3 produced significantly higher levels of gamma-IP activity (p(gammaEF)D-5) without adenovirus coinfection.
70 times). p due to adenovirus co-infection,
Gamma-IF' activity was decreased. To determine whether VA RNA mediates the increased expression of gamma-internene, plasmids)'pQ2 and p(gamma-IP)D-6 were incubated with psVOd or pVAs, respectively.
It was mixed with VOd (a derivative of psVOa containing the adenovirus VA gene) at a ratio of 1:1 and transfected into CO8 cells. 48 hours after transfection,
The samples were subjected to gamma-IF assay. p (Gammae F)
D-(', in contrast, showed a 2-3 fold increase in activity when co-transfected with pVS ■Od.

pLss showed a more than 10-fold increase upon co-transfection with pVAsVOd. Finally, pQ2, which contains the MA gene, has a high gamma-IFl value when coexioned with pVASvOd. These results indicate that the presence of the VA gene in trans is essential for adenovirus late m.
m containing the majority of tripartite leaders from RNA
Facilitates expression from RNA.

The decrease in gamma F activity due to pQ3 superinfection observed in Table 1 may be due to the toxic effect of adenovirus infection on cell metabolism or changes in adenovirus late mRNA.
This may be due to competition of transcripts from DNA genes.

The results of the gamma interferon activity patch assay were also supported by both immunoprecipitation and protein plotting methods (data not shown).
It was shown that gamma interferonase was expressed.

These results confirm that VA RNA responds to increased expression due to adenovirus superinfection. Increased translation by adenovirus MA'RNA was tSV
Increases expression of chloramphenicol acetyl transferase from the 40 early promoter. 5V4
Co-transfection of a plasmid containing the chloramphenicol acetyltransferase (cAT) gene under the control of the early promoter and a plasmid containing the adenovirus VA gene. compared to co-transfection with a plasmid lacking 5-J
, obtains a 0x higher level of CAT7g properties. RNA plot analysis showed that CAT mRNA was not increased upon co-transfection with the MA gene, so the increased expression was due to an increase in transfection. Under the control of the SV40 early promoter, expression of the plasmid containing the cellular xanthine-guanine phosphoribosyltransferase gene is also increased to the trans7 region of plasmid 9 containing the VA gene.

Similarly, expression of other atypical proteins, such as tissue plasminogen activator, can also be significantly increased using collateral DNA, such as the VA gene.

pL58 and pQ were introduced into monkey CV1 cells by co-transfection with plasmids encoding the SV40T antigen.
3! A similar increase in translation efficiency is observed when

This increase in VC1 translation efficiency is not limited to CO8 cells. Furthermore, internal and minute %J? , (D
Translation of both proteins (HFR and gamma interferon) can be stimulated by 7 VA RNA. Gamma-interferon cDNA recombinant (pQ2) T-transfected cells were transfected 72 hours after transfection.
It produces interferon at a level of 1 μg/1061 fl cells. This is a very high level of expression assuming a translation efficiency of 5-10%. Finally, the increase in translation efficiency is D
Not unique to HFR and gamma-interferon, both human interleukin 2 and human proinsulin are also efficiently expressed in similar vectors.

Table IA A, 7 virus co-infection? associated trans7 protein prIF-6'-1420970560pL58
1-3 1.690 6.670
2.920pQ, 2 + 1-3
15.200 11,550 26.320pQ3
+-1-311,55011,5
5034,640B, co-trans7ex/yomp9xmi) with MA gene psVOa
pVAsVODprIF-63,8508,770 pL58 2,230 26.320T
) Q, 2 8,770 20.000 Table IA legend γ-interferon activity in CO8 cells transfected and infected with adenovirus A, CO8O8 cells water displaying plasmid) transfected with DNA as previously described. Cells are subsequently infected with adenovirus or mock infected.

At the time of adenovirus infection (36 hours post-transfection) and 18 hours post-infection (5 hours post-transfection)
Samples are taken for gamma interferon assay at 6 hours). Is it active as mentioned before? Determine unit/rR
Expressed in 1/106 cells.

B, Plasmid displaying CO8 cells transfected with 2u/g pSV○d or p
60 hours after transfection, samples are taken for γ-interferon assay.

Transformation vector pAdD26sVpA (3) is Kau
fman et al. i) Mo1. Ce1l Biol,
2 (Ill: 1304-1319 (1982)). It has the structure shown in Figure 3. Briefly, this plasmid is an adenovirus 2 (Ad
2) Mouse dihuman acid reductase (DHPR) cDNA under the transcriptional control of the major late promoter
Including genetic information. The 5' splice site is adenovirus D.
The 3' splice site contained in the NA (derived from the q-munoglobulin gene) lies between the Ad2 major late promoter and the DHFR coding sequence. S
The V40 P77 polyadenylation site is located downstream of the DHFR coding sequence. pAd-D26S'VpA f
The prokaryotic-derived part of 3) is from psVOd (Me
llon, P. , Parker, V.

GltxZma, n, Y, and Maniatis
, T, I 981, Ce1127: 279-2
88) p, which is known to inhibit replication in mammalian cells.
Does not contain the BR322 sequence (Lusky, M. Botchan, M. l 981, Natur
E(Loncol. 293: 79-81).

pAdD26sVpA (31 is converted into plasmid pTPL as illustrated in Figure 3. I) AdD26sV
One of the two Bs tI sites in pA(3)? Deleted pAdD26SVp/(31w -! 9 SumiV
pAaD26sVpA (31(d) K conversion f le,
This was partially digested with Pst,H (resulting in a subpopulation of linearized plasmids with only one PstI site cut, which can be used for those with insufficient enzymatic activity) and then treated with Klenow. Then, connect it to Plasmi r? This is achieved by recircularization, transformation into canine coliform bacteria, and screening for those lacking the P site located at the end of the 5y40 polyadenylation sequence.

Three adenovirus leader and virus-associated genes (VA genes) are shown in Figure 3.
pAaD26sVpA(3Xd) (c insert. 1st
pAaD26sVpA (3) (d) %' Pvu
It is cut using an I-technique to form a linear molecule open at the first 3' portion of the three elements consisting of three separate leaders. pJAW 43 (Zain et al., 1979.Ce:sl 6851) was then digested with KXhoI and Klen
ow, digested with PVuI, and electrophoresed on an acrylamide gel (in 6% Tris-borate buffer; Ma
Niatis et al. (1982) supra) isolated a 138 base pair fragment H containing the second leader and part of the third leader.

138bp7 lacment f Pvu engineering I digested pAdD2
6sVpA (3) (d). The ligation product (E. coli?) was used to transform into tetracycline resistant colonies using a Grunste 7 nucleotide probe that hybridized to a 140 base pair fragment.
ln-Hogness method? Screening using DNA from a positive hybridized colony was extracted, and the 5' or 3' of the inserted 138ta site was a PvuI site specifically rearranged into the third adenovirus late leader. Test to see if it is above construction. The upper direction of the PvuI site is at the 5' end of the inserted 138 base pairs. This plasmid is referred to as pTPL in FIG.

Digest the SV40 DNA with AvaI and add XhoI linker? fragment and digested with XhoI to create Xh
I) 4th largest (D) fragment by gel electrophoresis. By isolating SV4
SV40 Ava engineering ID fragment containing the Q enhancer sequence. Insertion of the Lincoln D fragment results in a single direct repeat sequence of the SV40 enhancer. This is rAdD26Sv in conjunction
The results are proportional to the D fragment of pA(3) with an XhoI linker attached. pCVSVL2-TPL
The orientation of the SV40D fragment within is the same as that of the SV40 late promoter, as is the adenovirus major late promoter.

pCVSVLz-TPLK7te/! Accompanied by Filth (V
To introduce the AMt gene, first, a plasmid pBR containing an adenovirus type 2 Hind engineer IIB fragment is used.
322% - constitute. Adenovirus type 2 DNA'l;
(B fragment is isolated after digestion with HindIII and gel electrophoresis. This fragment was previously digested with HindIII.
Digest with II and insert into pBR322.

After transformation of Escherichia coli with low resistance to Amugi 7, the recombinant was screened for insertion of the fragment using HindI, and the direction of insertion was determined by restriction enzyme digestion. pBR3
22-AdHindA contains a fragment from adenovirus type 2 HindI in the orientation depicted in FIG.

As shown in Figure 4, plasmid) pBR322-
VA gene from Aa H1nd Ae, HpaI
Digestion by P2coR addition of linker and P2c
It is conveniently obtained by digestion with oRI] and recovered as a 4 kb fragment. The fragment with EcoRI cohesive end 7 is called pTPL (previously labeled with EcoRI
(digested with ) and ligated into the KcoRI site. After transformation of E. coli HBIOI, selection is made for tetracycline resistance, and colonies are screened by filter hybridization to a NA probe specific to the VAA gene.

DNA is prepared from positive hybridized clones and confirmed by restriction enzyme digestion.The generated plasmid is p
It is called 91023.

Exclude two EcoRI dissections within p40023. p91
023 is completely cleaved with EcoRI, generating two DNA fragments = one is AdMLP, one is TPL.
, DHPR.

SV'40pA site, S'V40 Aya■I fragment, pBR322'ret gene and replication origin (no toxic region) about 1 in 7 fragments, and the others are vA cogenes? This is a 1.3 kb fragment containing .

Klenow of the terminal '4PolI of both fragments
Fill with fragments, both fragments (beat, 1
．． ．． 3kb and 7kb) are religated together. 91023 as well as MA
The Grunstein method used for the VA gene contains genetic gold, but the two ECORI sites are missing.
-Identified by Hogness screening and conventional restriction site analysis.

A single ps tI site in p9+o2a (A)? Except,
Replace with Ec OR engineering site (Figure 5). p91023
(A) Completely cut with Psd and K of Pol
treatment with lenow fragment to generate blunt ends. EcoRI! J car 5p91023 (A)
ligates to the cut Pst site.

Linear p91023(A) (EcoR at the cleaved Pst site)
I linker is attached)? unbound linker
, digested to completion with EcoRI, and religated.

Plasmid “1)91023(B) was recovered, and I)9
Structure similar to 1023(A)? It is identified that having
An EcoRI site is located in front of the Pst site.

Plasmid p91023 containing the DCORCOR engineered product gene is American Type.
Culture Collection.

Rockville, Maryland, E. coli p.
It has been deposited as csF'-1 under ATCC number 39754 and is available.

Example 4 Translation activator (adenovirus VA; l!i gene) described in Examples 2 and 3? The usefulness of vectors containing?
Human antithrombin A II in both transiently transfected and stably transformed cells.
(ATIII)? This will be demonstrated using the encoded cDNA.

Human antithrombin I II cDNA DNA-
``Physiological inhibitors of coagulation and fibrinolysis'' (D.

Co 11en, B, Woman and M, Ver.
Edited by stvaete.

Elsevier, Amsterdam, 43-54”
:-TJ, Peters, en, G, D in
udek-Woj ciechowska,.

L, 5ottvup-Jensen, and s, Mag.
Human antithrombin I protein full-length C was isolated from a human liver cDNA library by hybridization to a specific oligonucleotide probe deduced from the amino acid sequence by Nusson et al. (1979).
Isolate the DNA.

A human liver CDNA library was prepared using a conventional method (Maniati).
Prepared by S et al. po 1yA+mRNA7 isolated from human liver and treated with methylmercury, the first cDNA
It was performed by reverse transcription on DNA. After treatment with bases, second strand synthesis is accomplished with the Klenow fragment of DNA polymerase, and the double-stranded cDNA is subsequently treated with Sl nuclease, EcoRI methylase, and the Klenow fragment of DNA polymerase to obtain terminal ? disconnect. FCOR Nirinka? In addition, excess linker n E
Removed by coRI digestion and passed through an 0L4B column. c.
DNA%'? 10, packaged and plated in vitro (C600hfl, RonDa
vis, 5tanford University
high-frequency lysogenizer obtained from ). Total so o, o o
amino acids 243 to 248 (Met-M6t
-Tyr -Gln -Glu -Gly) and 30
4 to 309 amino acids (Glu-Glu-Me
Screen with two sets of oligomers prepared from t-Met-Leu-Mal). Based on the degeneration of the amino acid code, one set of oligonucleotides consists of 8 different 17-mers and the other consists of 16 different 17-mers.

5 pq 7ml Contains denatured sperm DNA-%-5
XSSC.

Hybridization in 0.5% SDD and 5X Denbart solution? implement. Thirty-eight positives that hybridized to both probes were identified, plaque-purified, and isolated! It is identified by rehybridization to nucleotivib.

During the course of these experiments, S. C. Bock et al.
82) (Bock, S, C,, Wion, L, ,
vehar, G.A., and Lawn, R.M., Nuc.
, Aclds Res, 10, 8113-8125) published the nucleotide 9 sequence of human ATIII. Therefore, to isolate full-length clones, the initiator ATG%-surrounding 17-mer ori! Nucleotide (TATrC island A'1Gff)ATAGG:)2 was synthesized and hybridized with 38 positive ones. 13
One of the positive samples was prepared and lambda-ATIII
-03 (named AT3-(3+, it is about 1.4 k
b Inserts are included.

Insertion of ATI cDNA into U current vector The expression vector used for ATII expression is pq2 (Figure 2(c))
was derived from. Digest with pq2'l Ps tI to remove -f interferon cDNA Y and isolate the 7.5 KB fragment from agarose gel cATIAT
Does the III cDNA have an internal EcoRI site? Because it does not include.

It can be cleaved by EcoR digestion, and 1 is separated from lambda-AT3-O3 by agarose gel electrophoresis.
．． A 4KB fragment is isolated. Pre-synthesized adapter two Pstl 5'GGCGAGCCTG3' Ec
Insert the EcoR engineering ATI engine into the oR engineering ACGTOCGCTCGGACTTAA vector. Addition of the synthetic adapter is accomplished by yIJ oxidation only at the 5' end of the 10-mar. This reduces the packaging ground of the transformants.
Leave the R end. Excess adapter by low melting point agarose gel electrophoresis? After removal, the 7.5 KB fragment was extracted and ligated to the ECOR A'I'I IC DNA fragment. The ligation mixture was used to transform the large intestine [HB101'f tetra-length icrin resistant and ATII
I cDNA 7: ff-coded T4 end label (Bn
Glund, P.T. (1971), J.B.C.
246°3269-3276) cDNA fragment? The Grunstein-Hogness method (
1975, PNAS, 72°3961-3965) to identify positive substances. Two clones were obtained, p91023ATI: [-O3 is ATII
I cDNA 5 contained in the appropriate orientation with respect to the adenoviral promoter and p91023ATIII-El
Contain cDNA 7 in the opposite orientation.

Plasmid” p91023 ATI AnyC3 (pA
TII knee 03) (Fig. 6) is AmericanTypOCgl, ture C as a strain of large@M-HBIOI.
o11ection (Rockville, Ma, ry
Land) under commercial number ATCC 39941.

available.

p91023-ATIII-C-3 and p910
23-ATE"-El is a PBR322-derived iM-derived product lacycline resistance gene for reproduction in E. coli and sv4 for replication in CO8 cells.
Q derivatives and pBR322 “poison” region 1. IKB
Deleted (Lnsky, M., and M. Botcha
n (1981), Nature.

293.79-81). cDNA in cos t le cells
The activation element for the expression of A is the adenovirus major late promoter (AdMLP; SV40 enhancer (
cDNA copy of the adenovirus tripartite leader; 5' splice site from the second intron of the tripartite leader and 3' from the mouse immunoglobulin gene
Hybrid indone consisting of splice site (
Kaufman, R. J., and 5harp P., A.
(1982) (Mo1. Ce1l Biol, 2
, 1304-1319); SV4o? 7J phase region polyadenylation signal; and adenovirus VA gene. Human antithrombin T-enin encodes 1, 4
The direction of Kb cDNA is shown. p91Q23-ATL
: Engineered C5 contains the ATIII cDNA f in the proper orientation for transcription from AdMLP.

CO8 cell expression As described in Example 1, cos+j+D into cells.
EAK-p91023-ATIII-O3 and p910 by text run-initiated transfection
23-ATIII-Etyr was introduced. 24 hours after transfection, cells were treated with serum-free Dulbecco
Wash 1 condition with modified Eagle medium after 724 hours of culture.

rabbit anti-human antithrombinae II (Robert
Rosenberg, MF3ssachusetts
ENG n5 titute ofTq chno ], o g
y, obtained from ) for radioimmunoassay. Alternatively, 48 hours after transfection, cells were labeled with 353 methionine (I mCi/mQ) for 4 hours. For conditional conditions, the culture medium was harvested and subjected to immunoprecipitation and SDS gel electrophoresis as described in Example 1. Prepare cell extract for transfected CO8
Table 2 shows the test results for ATI construction under cell conditions.

Table 2 AT proteins in genetically engineered mammalian cells were assayed by radioimmunoassay.

The HO stable strain is conditional with ATEE in the medium.
CHO-DUKX Bll, 0
21 nMCHO-B], 0neo
5.2 nMCHO-A5,0neo
4.5 nMC08M transferable infection p9l023-ATAEI-EJ, , 01
8 nMp91023-A'll' engineered C51,9
Chinese hamster ovary lacking DHFR due to 2nM dihydrofolate reductase (DHFR) gene (
cHO) For cells, use Urn-aub.
PNAS, 77, 4216-4220 ('
1982). plasmibi p91023
-ATEEI -O3.

psV2Neo (Southern, P., and P.
Berg, 1982 +J, Mo1. App1. G
enet,, l, 327-341) Work pAd
D26sVpA(3) (Kaufman, R,J,
and 5harp.

P, A, Mo1. Ce1l Biol, (1982
), 2.1304-1319) fX knee mixed 25μ9 p91023-AT engineering I knee C3, 2, 5
μF I) SV2NeO and 25μC/pAdD2
6sVpA (3)), sodium acetate CPH4,5)
'Up to 20.3 M and 25 volumes of ethanol? Add and precipitate. The precipitated DNA was left to air dry, and
J, Mol by Harp et al.
, Biol, Suio: 601-621 (198
2) and < 2 XHEBSS(,5
m)) (ch, u and 5harp Gene,
13, 197-202 (1981)) K resuspended 1
．． Mix vigorously with 25M Ca, C#z (-5m/').

Calcium-phosphate-DNA precipitates were pre-subcultured in 5XIO' cells/10 CrIL dishes in ink at room temperature for 30 min (241
1.1) Remove the culture medium of the cryo DLTKX-Bt cells and add to the DNA-calcium phosphate precipitated cells. After incubation for 30 minutes at Hefuchi,
Alpha medium (F'
lov La, bs) 4 and the cells were heated at 37°.
Incubate for 5 hours. Remove the medium from the monolayer of cells, add 2rnl of alpha medium 2 containing 10% glycerol for 3 minutes at room temperature (24°C), and wash the cells after removal.
0% calf fat J'I'I'I serum, 10μ each (J/ml
Seed in alpha medium containing thymidine, adenosine, deoquine, adenosone, penicillan and streptomycin. Cell TI after 2 days: lo% dialyzed calf fat It
'll Vi';'Nigiline and streptomycin? Including, nucleo-length ids and missing parts, , 1 rr
The cells were cultured for 311' passages at a ratio of 1:15 in alpha medium containing vy/meqG 418 (GI BCO). 4
-5 days later the cells are replated on the same selective medium J+1 (lacking nucleosides).

Colonies appear after 10-12 days of subculturing onto selective media.

Pool colonies (approximately 20) from each dish and use psV2
Coexistence of 0418 to maintain Neo selectivity 2? Increase the concentration of methotrexate.

7J) Maintaining psv2Neo while using ATI engineering
It is not essential for cDNA gene amplification. The initial pool of transformants was conditioned by radioimmunoassay of the medium or following 358-methionine labeling and immunoprecipitation (S
Is AT activity even if it is assayed by both DD-polyacrylamide gel electrophoresis? Not shown. ρ・1~while, 0
．． 02.01 and 1d AT after selection in medium containing 10 μM methotrexate can be observed with both methods (Table 1). After that, obtain 7 clones by dilution blating. AT produced in CO3-produced CHO cells■
Ae is active due to its ability to bind to thrombin, and binding was shown to be enhanced in the presence of heparin.

Example 5 mRNA of interest 7 isolated from Bowes melanoma cells according to conventional methods previously used in other cells.
t, 'PA f go 1' by reverse transcription from RNA
The cDNA and IlI gene were obtained.

tPA protein is Bov=eS melanoma, I'll
Cell strain (Rj, Dr. New York
Amino acid sequence analysis was carried out using a single piece of meat (available from K, University). The ascending N-terminus? one N-terminus contains three more amines I than the other.
G? include. These two N-termini (1) are glycine N-terminal (Glv-Ala-Arg-3er-Tyr-Gln
-Va11e-Cys -Arg -Asp -G
ln-Lys -Thr-Gln -Me wo-tech1e
-Tyr-Gln-Gln-His) and SeriyN-
Terminal (Ser-Tyr-Gln-Va11e-Cys
-Arg-Asp-GIn-Lys-Thr-G], n
-Met-11e-Tyr -C1]1-Gln-Hi
s ).

BO”F: Metsenger RN from eS melanoma cells
A1''-MIE and used as a template for the synthesis of cDNA.

cDNA 7. Cohesive, two @ 釧娀 produced. Tailing or V
Introduce the tetraticline resistance plasmid vector with a synthetic linker. A library of cloned plasmids (each clone containing a unique type of mRNA present in Bo'vt-e S melanoma cells) was isolated from canine coliforms in a female vector. Transform and prepare antibiotic-resistant E. coli cells? select.

CDNA encoding at least a portion of tPA
Grunstein and Hogné SS screening method (Grunstein et al., 1975. PNAS,
72.3961). In this method, DNA 7 is immobilized in single-stranded form and screened for its ability to hybridize or bind to a radiolabeled probe. Such probes are short DNA oligonucleotides whose sequence matches the amino acid sequence of a small portion of the tPA protein. The amino acid sequence of 15-20 amino acids from the N-terminus of tPA is used (Figure 7
It was shown to). Since some of these amino acids may be coated with one or more 3 nucleotide cobins, the following routine method covers all possible 17-mer probe pools.

Thereafter, Grunstein et al. until clones in the library were observed to hybridize to the probe
[CDNA primer screening was performed using the method of HOpnes S. Upstream sequence of primer? Determining partial dideoxy primer extension (Wallace et al.

Nuc, Ac1ds Rc, , , 9, 3647
-3656 (1981)), it is confirmed that the flown encodes a part of t, PA. Is this a codon that corresponds to the clone's tPA6) N-terminal amino acid sequence? Including 11? Demonstrate.

cDNA plasmid 1 observed in the clone
No.t41. released, radiolabeled, and sequentially cDNA
Other duplicated jPAcDNs observed in the library
Used as a probe to identify fragments of A and Z).

- mature 1c (excluding duplicate sequences of 2i),
This process continues until a fragment encoding the PA protein and portions of the untranslated 5' and 3' regions is identified.

2 different libraries - 2 screens together
, a cDNA clone covering the complete yl coding sequence of PA? It's number 1. CDNA clones saml and S20 have an asymmetric linker? The attached library ρ1 was isolated. This library was prepared using the following standard method (Maniatis et al., Molecular Cloning - Laboratory Manual - Cold 5pr
j, ng Harbor Labora, tories
.

Cold Harbor, N. Y. (1982)
).

That is, the CDNA from the Bawes 41 cell mRNA is ligated with an EcORI linker at the 3' end and a 5all linker at the 5' end, and the ligated CDNA is inserted into an appropriate vector.

cDNA (GC-tail)-”c
It was isolated from a DNA library (Mani*tis et al., supra). Figure 8a shows a cDNA consisting of a partially overlapping set of tPA complete coding sequences.
Clones Sam1.320 and Ea], y stones shown.

The technique used to combine three fragments into a single coding sequence (Fig. 81), schematically shown in Figures 8C and 8d; The construction of a plasmid for cDNA gene replication is illustrated. The composition of plasmid intermediate p5 is BO
Gene by Stein et al., 8.

17-24 (1979). 2u yeast plasmid 9 is a commercially available product. Is the “+” indicator a restriction enzyme site in the plasmid? show. Restriction enzyme and/or D
Klenow fragment of NA yp limerase (
The processing by the method (hereinafter often referred to as "Klenov") is indicated by the name next to the arrow. These enzymes are commercially available. Use common enzyme reaction conditions. Note that ligation of the digestion products of some restriction enzymes creates DNA sequences that are not hydrolyzed by other restriction enzymes. For example, Y
The construction of the Op4 plasmid eliminates the u HpaI site. Figures 8c and 8d show the construction of a plasmid containing full-length cDNA clones from the three cDNA fragments identified in the library. Illustrated. In Figure 8(c), the plasmid ()"Yop4k is cut with Xba, the ends are made blunt with Klenow, and the linear plasmid is cut again with EcoRI.
PA cDNA fragment (contains the untranslated 5' end portion? See boxed 5FIII site in Figure 7)
Digestion with 55alI, Klenov treatment, and EC0
Isolate from the plasmid by digestion with RI. Linear YOp4 and tPA cDNA 5' fragment S
amlTX: ligated to form a plasmid l9oi%-shadow by ligating the XbaI and 5alI sites l)
A salinized site is formed. Plasmid ° Engineering 901? Ligate with E. coli and screen for ampizoline resistance.

Plasmid l901 was treated as shown in Figure 8b and t
3′ fragment of PA cDNA (EEdl) (
plasmid) in order of Bgl engineering 1. Klenow and EcoR processing). Bg1
E site (indicated by a box in Figure 7) Plasmid ■9
01 to the C1,I digest. t, P.A.
cDNA of PstneyBg1 and EcoRI
- Discard the Pst engineering subfragment. The first one is because it is a non-coding area.

The second one is the third 7 lagmen) 820 (Fig. 8c)
6 because of the overlapped area! l), the discarded sequence is supplied by S20. Through these steps, plasmid)”J
118'Q obtained, it replicates in E. coli and is ampicillin resistant? Screen.

Plasmid Jl18'< Cut with EcoRI 1 Figure 8
2. Ligate with the central tpAc DNA fragment S20 shown in 0. The appropriate orientation of the S20 fragment in J205 is the asymmetric end, which is generally known in the art.

Confirm by nuclease digestion.

This tPA cDNA gene was derived from the previously published human tP
The sequence of A differs from that in the number of nucleotides, and one amino acid is substituted as shown in FIG.

The starting plasmid for this example is pAdff)26SVp
A(31(Kau fman et al., Mo1. Ce1l B
iol, 2(11): 1304-1319 (19
82)).

Is that the structure shown in Figure 9aK? have Briefly, this plasmid 9 is a vector of plasmid 9 that is under the transcriptional control of the adenovirus 2 (Ad2) major late promoter.
Contains the 0 folate reductase (DHPR) cDNA gene. The 5' splice site is included in the adenovirus DNA, and the 5' splice site derived from the immunoglobulin gene
The splice site is located between the Ad2 major late promoter and the DHPR coding sequence. 5V40 early 4
The readenylation site is located downstream of the DHFR coat sequence. The prokaryotic derived part of pAaD26sVpA (3) is psVOd (Mellon, P., Parker
.

V., , Glnzman, Y., and Maniatis.
, T, 1981°Ce1l, 27.279288), and the replication of mammalian cells? pBR322 sequences known to inhibit (Lnsky HM, s and Bat
chan, M., 1981, Nature (Lond
on), 293.79-81.

pAaDz6sVpA(3)fX: Figure 9 a K-Lj
pcVsVL2 K conversion 5V40J) NA w AAvaI digested with I, ligated to the fragment with an Xho linker,
-Digested by Xho-engineering and Xho-engineering part? The fourth largest (D) fragment was isolated by cleavage and gel electrophoresis to obtain fragment 7 from Ava processing of SV4 Q.

Insert the linker-attached D fragment as shown in the first step, and directly repeat the SV40 Enhanced-) to? obtain. This is pAdD26sV at the time of ligation.
pA[3] noffi,! : Xho-I IJ linkage fragment 7 proportionality results. pCvS
The orientation of the 5V40D fragment in vL2 is similar to that of the adenovirus major late promoter, as is the KSV40 late promoter.

pcVsVL2w Examples 5 to (7) J205tPA
Convert to plasmid pB2L2 by a three-step process designed to accommodate splicing at the @gene. First, one of the two ps tI sites in pCVSVLZ, 4PSd (with reduced enzyme activity, is used to obtain a subpopulation of linear plasmids in which only one ps tI site has been cut). Partially digested and deleted, treated with Klenow, ligated and recircularized the plasmid, E. coli? After transformation, deletion of the Pst site located 3' of the SV40 polyadenylation west 1' column was performed. Screen.

Second, the plasmid was digested with Bgl and Klen
Bgl! of the immunoglobulin intron of the E. coli transformant colony. Screen for those in which the I site (3' splice site in Figure 3α) is disrupted.

Third, digestion with PstI, K1enOW treatment, Bg1
Linkage of aerincar and excess (100 units/μgD
NA) BglII VC digested Psd site 4
Convert to BgI site. The resulting linear DNA y )
! J Soot - low melting point agarose in acid buffer (1,2%)
Isolate the rainbow by gel electrophoresis on a gel. This DNA is then ligated in 4 microns at a concentration of 1 μ9/ml at 24° and used to transfect E. coli (HBIOI Y) (see Haniatis et al., supra). Tetracycline-resistant colonies were grown, DNA was prepared, and DHFR was digested with BglII and PvuI.
Existence of Bg]■I site at the 5' end of cDNA? Analyze,
Digest with ps t,I. DNA preparation on a large scale was carried out in C8C1 by tying DNA twice with 7 strings.

pJ205 from Example 5 was digested with HindIII and Sal, treated with Klenov, BamH
I linker? Concatenate T:.

The generated DNA was extracted with phenol and extracted.

Extract with form and extract with sodium acetate y 0.3 M (P
H4,5) and 2.5 volumes of ethanol? Add to serve as ethanol precipitate. Centrifuge it! 1llD
NA asked once, -? Let dry. Resuspend the pellet and add Bqm to sever the BamHI linker.
Digest with HI (1001' position 7 DNA/'!?).

Apply the digest to agarose dal and identify the 2, J, and kb bands. Collect the band and use lomM tryst](
Add an equal volume of buffer containing J(,7)H7,4) and 1mM EDTA and heat at 68° for 15 min to obtain DNA.DNA fphenol extraction L (2X), chloroform extraction C (2X), acetic acid) l) rum (, pH 4
, 5) to 0.3 M and addition of 2.5 volumes of ethanol K, 1 diethanol precipitate.

As illustrated in Figure 9h, the vector pBzLz is
II and treated with alkaline phosphatase,
Extracted with phenol (2X), chloroform (2X),
Na0Ac (pH 4,5) up to 0.3M and 25
Jetanol is precipitated by addition of 200 mL of ethanol.

This precipitated DNA is ligated with a 2.1 kb fungment coupled with a BamH linker from pJ205.

Ligated DNA) lx used for transformation of canine coliform HB1oi. Tetracycline resistant colony Kaoru Grunst
ein-HOgne! According to the ES law! 7 tPAcD
32p labeled probe specific to NA? Screening using , 7205 TX: HindIII and 5
Digested with alI, 32P-ful77-d, cTP
% and labeled with T 4 DNA polymerase to produce Prozo. positive hybrid clone ρ)
DNA was prepared and subjected to separate restriction enzyme digestions using HindIII, PvU, and Sac.
Screen for the presence of AcDNA. These digests demonstrate not only the presence of the t,PA cDNA, but also the orientation of the cDNA relative to the promoter within the lid. pLDsG is a plasmid containing tPA7 in the proper orientation.

Example 7 Co-transfection and amplification-I'ras,? )” pLDSG Oj HippAd
D26sVpA (31 (Example 6)?-5
0PgpLDsG and 0,5μ9pAaD26SYp
A (3), Na0Ac(7)H4,5 at 0.3 Mj
) and 2.5 volumes of ethanol.

The precipitated DNA was left to air dry, and 2X HEBSS (0,5
msu(chn and 5harp Gene, 13
Kaufm, 197-202 (1981)).
J, Mol by an and Sha, rp.

Biol., 150, 601-621 (1982).
Mix vigorously at 5 m/liter. Calcium-phosphate-DN
A. Leave the precipitate at room temperature for 30 minutes, then CHODU
KX-Bi II:III cysts (chasin and U
rlaub, P, N, A, S,, 77.

4216-4220 (1980)). Growth retention of these cells has been previously described (Kauf
man's work 5harp, J, Mo1. Biol, supra, and Chasin and Urlaub, supra).

DUKX-B l thin JFa are subcultured in 5XIO/1ocTL dishes 24 hours before transfection.

Culture medium? Remove the DNA and add to the monolayer of cells in a phosphoric acid precipitate. lO% calf fat serum after pencubation for 30 minutes at room temperature? Alpha medium containing (F'-1o
W) Incubate smlf-added cells at 37° for 4.5 hours. Remove the medium from the monolayer of cells and add 2-alpha medium (F'low) containing 10% glycerol (
Add cells for 3 minutes at room temperature (24°) and remove cells. Wash, 1
0% calf fat serum, 10 μy each of thymidine, adenone, and deoxyadenosine are inoculated on alpha medium containing Nigilin and streptomycin. 2 days later,
10% dialyzed calf serum, inizolin and streptomycin? Subculture at l:15 into alpha medium containing but lacking nucleocyton. Cells after 4-5 days? , again seed on the same selective medium (lacking nucleoside).

Colonies that appear after 10-12 days are subcultured onto selective medium. Methotrexe (MTX) Selection and Amplification 2
I followed the diagram. In the first scheme shown below in Table 3, a single independent cloned transformant is isolated based on the incorporation of exogenous DNA (selection gene), and each clone is subsequently isolated with the product gene. operating under conditions that increase the expression of methotrexate (ie, growth with increasing concentrations of methotrexate).

In the second scheme, a pool of many independent transformants based on the incorporation of foreign DNA (selection gene)? Isolation and expression of product genes? Move under conditions that increase (
i.e. the concentration of methotrexate? growth). Individual clones from the population 1) iF1~, expression of the product gene? analyse. Clones exhibiting the highest levels of product gene expression are then grown again under conditions that increase product expression (ie, the concentration of methotrexate in the culture medium is increased).

The results according to Scheme 1 are shown in Table 4. Individual clones that can grow on alpha medium without nucleocytes 9 are selected, propagated, and assayed for tPA activity.

Activity was recorded in CTA milliunits/cell/day or mU/cell/day. (hereinafter fX: 9° tPA activity active Ketalone LL: I followed by 002μMMTX, o, 1f1
Selected for continuous resistance to MMTx and 0.5 μM MTX.

Clone 4C1 had no increase in tPA activity under MTX selection. However, when cultured in the absence of MTX and generated, Toop clones (H3B and BxoA) co-amplify and express a high percentage of tPA when selected for MTX resistance. The method here? Without wishing to be limited to a particular hypothesis, it is possible that this is due to segregation of the DHFR gene not linked to tPA in the sibling DI'JA from the original 4C1 transformant.

Subclones containing a single DHFR gene linked to the tPA selection gene were amplified along with the DHFR and tPA genes by MTX selection.

Table 3 Pathways for maximizing the expression level of tPA Pathway 2 Table 4 tPA in clones selected by NTX resistance
Activity (mU/cell/day) MTX concentration (μM) ri0-7' 0 0.02 01 051
E2 0.2 0.015 0.005 0.0
054D2 0.015 0.0015 0.003
0.0022D5 0.01 0.04 0.
04 0.0254 Cl o:o 4 0.
03 0.35 0.03H3B O,09
0,261,0 BIOA O,0250,090,19H8B
O,020,0450,035H12B O,050
,150,065D8B 0.08 0.095
- The results for route 2 are shown in Table 5. Nucleondo? Including 1
Approximately 300
colony? A pool with (colonies from selection plates) was prepared. These transformants are 0.02/IM
These transformants were selected by sequential resistance to MTX and 0.05 M MTX. The original pooled transformants had tPA activities of 0.03, 0.9 and 1.9 mU/cell/day for culture in medium containing 0.002 and 0.5 M MTX y, respectively. However, individual clones within the population expressed tPA2 at the amounts shown in Table 5. Individual clones expressed PA at 50 times the range from 0.2 mU/cell/day to 10 mU/cell/day and varied by 4. Is this pathway a large amount of t, PA activity? This is preferred for quickly identifying expressing clones. Is route l combined with route 2 a more proliferative transformant? It is clear that it can be made.

＼, Table 5 ↓ 0.02M By growth in MTX 0.9
Selected cells ↓ 1.9 by growth in 0.05M MTX
Selected cells ↓ Clone #3 in 0.05 M MTX 4.25 47.0 93.5 12 6.25 13 1.55 16 0.2 18 0.3 20 10.0 Monitoring of tPA expression jPA 7 Monitor t, sensitivity test for the presence of PA! -j: Conversion of plasminogen to plasmin in the presence of fipurin? catalyzed. Plasmin is detected by the release of 125■-fibrin fragments from 125■-fibrin ρ, et al., which are immobilized on plastic plates (Stricklond et al., J.).

Biol, Cbem, 251 5694-5702 (1
976) or detected by degradation of chromogenic substrates (Drapier et al., Biochemie 61
, 403-471 (1979). The yf4-favored chromogenic substrate (referred to as S 2251) is Kabi (Diag
nostics, InC, Greenvhicb
.

CT). These assays are well known to those skilled in the art and are described by Astrup et al., supra, and Drapi et al., supra.
It is mentioned in er et al. tPA activity in serum-free medium: 11
! ! Plate (b) with cells in 5 ml x 105
cells/10CrIT plate)? It was measured by rinsing 9 ml and adding 4 ml of serum-free medium. Cell 3
Incubate for 20 hours at 7°C and incubate the temples in conditioned medium. It was taken out for testing. Activity measurements were expressed in mU/cell/day. In testing under these conditions, 13o
'vre S meginoma cell line is 0.02mU/cell/day? produced. h) t, the specific activity of FA is 100,
000 units/rv. The rate of degradation of 125 nefibulin or 82251 is directly correlated to the amount of tPA that catalyzed the conversion of plasminogen to plasmin.

Samples of conditioned media from cells deficient in tPA expression are negligible for these assays! Only tsukground occurs. This negligible background is due to activated non-fibrin protease. This is because the background in the chromogenic substrate assay does not change from the standard assay even with the removal of fibrin.

In contrast, the activity from the iPA-producing CHO cell line is very similar to that exhibited by Iowes melanoma tPA? show. Determination of tPA activity is obtained by comparison with a standard curve for urokininan (Lea Pharmaceuti
cals.

is lugie). Units of activity (cTA; Committee on Thrombolytic Agents) have been determined by comparison with the WHO reference standard for urokinase.

t, PA synthesis was performed in single cells (roughly 2xlO/10)
Wash 5 ml of methionine twice with non-sinking medium and monitor by adding L ml of normal methionine-free medium containing 1 mC135S methionine. Cells are incubated at 37°C for 4 hours and prepared. The culture medium was assayed by immunoprecipitation with rabbit anti-human T,PA using Staphylococcus aureus as an immunoadsorbent.

According to the results obtained by gel electrophoresis of all the labeled proteins secreted from colony H3B.

Original H3 grown in medium without nucleosides
, B subclone and 01 pM in MTX (7) , i!
, i 9jrl VC, lζ) The only major difference in secreted proteins between 13B cells is 67.
There is a strong kana band that migrates to 000 gluton C.

This band was specifically immunoprecipitated with the rabbit anti-PtPA antibody and was similarly prepared from the EOWeS melanoma cell line.
Ru. Are these results resistant to O1μMMTX? H to have
It is shown that the expression of 5 human tPA increases 10-fold upon selection of the 3B clone.

Example 8 Construction of a flanking linked vector This example demonstrates the stop codon y! of tPA! This figure describes the construction of a transformation vector directly linked to the start codon of 1''DHF'H.
LDsG empty tPA 3' O L U DHFR5'
Non-conversion W area? It is to be removed. This example is M13
It is based on the use of DNA oligonucleotides 9 to simulate DNA synthesis from phage templates (Wallace et al., rscience J209:1
396 (1980), ZO11er et al. Method
in Enzymology Vol, 100:4
68-509). See Figure io.

pLDSG i;l: digested with BamHI, 4
．． The 5 kb fragment F was isolated by pneumophoresis. Is this region the majority of the non-pBR322 region of pLDSG? Contains.

Digested with phage M13mp8γBamH1. Then 4.5 kb of linearized phage Q pLDSG
Combined with fragment, M13 construct? Host bacteria (JMt
Used for transformation of 03). t, 32p-labeled probe fused to the PA gene? Use Benton-D
avis's 1-3cjance J l 96 (198
Plaque screening is performed using the method described in 0). Clones that align correctly are propagated to produce a copy of DNA 7. t, the correct orientation of the PA gene is determined by restriction endonuclease degradation (BamHl, EcoRl and Pv
ull) The amount for ¥ can be determined using known techniques.

DNA is isolated from plaques that exhibit the correct orientation. This single strand of KJ DNA can be used in conjunction with a synthetic primer that bridges the region of the phage to be removed.

The function of the primer is the 5' and 3' removal region D.
It is fused to the NA, thereby looping undesired misplaced DNA tags and thus preventing the NA from acting as a temperate in the subsequent primer extension step. In Figure 10, the 5' and 3' ends of the primers and the M13-
The corresponding positions in the LDSG are respectively named B and A.

The primer is made by chemical synthesis and is DHPR-3'
t at the amine end of the PA gene-TAA-AT
5' of the 9 translated nucleotides 9 following the G-contamination
- Consists of the terminal 9 translated nucleotides.

Mix phage DNA and primer r, primer 'r4 DNA ligase and DNA #? It is extended by the Klenow fragment of S. limerase.

This is generally explained in Wallace et al. and Zoller et al., supra. The product is Ju103%-
used for transformation. The replicative phage DNA from the second primer was fused to the 32p-labeled primer,
Undesirable area by Sanger? It has been confirmed as the removed dideoquine nucleotide 9 sequence (S
Anger et al. Proc, 1'Jat1. Acad, Sc.
i, 74 pp 5463-5467 (1977)) - which is degraded with RamHi and isolated by electrophoresis. This fragment was separated from the gel and pL obtained from BamH1 digestion as described above.
DSG (7) combined with the 2.5 kb BamH1 fragment. The ligation product was used to transform E Co11 HBIOI to create a tetraniphrin-resistant clone?
identified, made into plasmid rDNA, and specified. 2.
The correct orientation of the 5 kb and 4.5 kb fragments is identified as P2 smid resistance, LD GL.

ko (D 7' 5 sumi)'i'j: pLDsGoI
pAdD26E3VpAI3) can be used as the linked vector in Example 7 instead.

Example 9 pLDsG ld is digested with BamHl and a 4°5 kb fragment is isolated as shown in FIG. 1
Virtually all tenopBR3224n was excreted along with a 2.5 kb fragment by 3amH1 degradation.

pAdD26sVpA (3) is similarly degraded. t,
75 y'up containing PA and DHFR genes
LDSG k L U pAdD26SVpA (31) is used for CHO cell cytoplasmic transformation as shown in Example 7.

The method of this embodiment is illustrated in FIG. ii.

To construct a vector containing the adenovirus tripartite leader, I) start from B2L2, which
Cleaved by Pvuee to form a linear molecule? create. Then,
pJAW43 (Zain et al. rcellJ 16,851
(1979) digested with %"
Isolate with 138bp fragment followed by PvuI
I decomposition pBzI, binds to z. Combined product yEcol
i'IX: probe used to transform to tetra-length icrin resistance, labeled with 32P and fused to a 5140 base pair fragment? UseGrunst, ei
Screen by the n-Hogness method. Pv created and regenerated from the colony ρ) that fuses exactly
Test whether the ulI site is 5', ρ, or 3' of the 138 base pair DNA inserted specifically into the second and third adenoviral post-leaders. In the correct direction of Pvu Engineering I site.

138 base pairs would be inserted on the 5' side. This plasmid is named pB2L2-TPL.

Adenovirus-associated (VA) gene w pB2L2-T
Adenovirus type 2 DNA for insertion into the PL
Decompose with 汀・I 5 Hj, ndIII. After gel electrophoresis by a known method, the E fragment was isolated, this fragment &K] was filled with 6 cylinders at 10 W,
, resolved with HpJ, coupled to an ECORI linker (cooperative), digested with ECoRI, and the 1.3 kb band separated from a pugarose gel. This fragment 7
I) Binds to the FEcoR esite of B2L2-TPL, which has been previously digested with ECoRI. , E Co11 HB
After transformation of the IOI and selection for tetracycline resistance, the VA gene VC1p:1 is screened with the filter IAIIj for the heterologous 1 (DNA probe).
Y crop 1 clone, identified by restriction endonuclease digestion. The resulting plasmid was digested with pBzLz-TPI7. This is suitably used in place of pBzLz in Example 6, and the modified pLDsG product '.
) should be used with

Example 11 The vector psor has been described (Gillies et al., rce11J33 near 17'-728 (1983)).
This is a psV with the SV40 enhancer removed.
20PT no 84 (5phl from Pvu engineering,
base pairs 64-270).

Mulligan et al., rscj, euceJ 209:
1422-1427 (1980). This DNA becomes resistant to mycophenolic acid when cells are grown in the presence of xanthine and hypoxanthine. −?Containing I kb fragment'/pser Eco
It was inserted into the R site and pserx2/35 was induced.

This vector has one BamHl: site y. This was decomposed with BamH1, treated with basic phosphotase,
Then ligate to the pLDSG fragment. This fragment is generated by digestion with BamHt and separation of the large (b), 5kb) fragment as described in Example 8. The ligated DNA was used to transform E. coli HBIOI, selected by ampinlin resistance, and colonies were 32p Keyaki-identified,
The presence of the ipA gene by hybridization to the PA probe is screened by the Grunstein-Hogness method. This probe is J205 'If
< Disassembled with Hind Eeeee 5all and 32p-α
- dCTP and aTTP containing no radioisotope,
It can be made by standardizing RT) NDA with T and l DNA polymerase in the presence of aATP and dGTP.

A positive cross (11 clones were propagated, 7 plasmids were recovered). Figure 12 is the lid (p7B1).
)'r1 I'm happy. This DNA was introduced into myeloma 41J558L as described in G11lieS et al. supra, a modification of the 5andri-Golclin method of protoplast fusion. After 36 hours of protoplast fusion, &(n cells'15 mfl/ln, l
Mycophenol IL was grown in medium containing 250 μq/ml xanthine and 15 μq/ml hypoxanthine X, and cells that had incorporated plasmid v DNA were selected for resistance. One clone isolated by this method was t,
PA was expressed at a rate of 20.0'5 mU/cell/day. Other suitable parental cells are ATCCCRL1580-i
rs is another non-secreting myeloma cell line.

Example 12 Transposition activity transcriptional actinator EIA, adenovirus early region 2 promoter? For use with vectors containing

, CVSVL is Kanfman et al. rMol, Cel'
, Biol, j '2 (Il11304-1319(1
982) K disclosed. pCVSVL'YBal
, treated with I(:Lenow, bound to the Xhol linker (cooperative linker), degraded with Xhol,
The 4.2 kb fragment was isolated by gel electrophoresis.

Adenovirus 2 DNA 5 Decomposed with EcoRl.

EcoRI F+ fragment (map unit 707
~75.9)% - recovered. F fragment 7:'x
Decomposed by h○1 and E2 promoter subfragment? Isolated by gel electrophoresis. Is this subfragment FCORI and Xhol sticky end? The 4.2kb fragment of pCvSvL obtained above
attached to the oRt and Xhol ends. Is the resulting plasmid a bacterium? used for transformation. Clones that hybridize to the standardized E2 probe should be recovered as pE2-7. pE2-'7 is a DHFR cDNA gene that utilizes the adenovirus early region promoter. p
E2-7 was constructed by R, Kingston and P.

5 harp method is given. Mol. Ce
1lBj, ol,, Voko, 4. pp1970-1
977 (1984)'& see.

pE 2-7 was partially degraded with Pstl and Klenow
treated with BgIII+), bound to a BgIII+) linker, and
It will be disassembled by g1 ■ engineering. This allows the tPA trap gene from pJ205 (Figure 9b) to be inserted into pE2-7 in a conventional manner.

The part of pJ205 containing tPA was excised from pJ205 as shown in Figure 9b, and the BamH1 sticky end',
<It binds to the BglII cohesive end of pE2-7 and binds to the 12th
(b) The plasmid Mpg2-7pA shown in the figure was obtained. pE
Is 2-7PA the adenovirus early region 2 promoter?
The t used is a direct transmission of PA. The EIA gene engineered promoter is obtained as a Kpnl fragment of adenovirus 2 (10~5.8 map units).

pE2-7PA, pFE2-7 and EIA gene vectors were used in a molar ratio of 20:1:4 for CHO cell cytoplasmic transformation as further disclosed in Example 7.

The transformants thus isolated were engineered to have 0 tPA activity.
．． QO8mU/gun/day is produced. Transformant obtained by omitting the EIA gene (1 at a ratio of 20:l) E2
-7 wives I)E2-7PA) (summer crab 0.000
3mU/cell/day? manifest.

Growth and selection under optimal conditions for tPA synthesis) results in cells with a different morphology that are unable to adhere to tissue culture dishes.

A pool of 300 transformants grown in nucleotide-free medium (engineered as described in Example 7, Table 4) containing low levels of tPA (0.01 mU/cell/day) produced A flat morphological feature? It is a CHO cell with When selection 2 was performed as described in Table 4 for growth in MTX below 0.05 μM, tPA levels were increased by 0.5 mU/cell/day and morphological changes were evident. . Is this kind of change more than 1.2 mU/fine, gun/day? It is also evident in producing clones (eg clones 9.12 and 20). These cells do not adhere to tissue culture dishes and therefore require higher levels of tPA.
Selection by expression is difficult. This can be achieved by adding (0.5-5% VAJ) to aprotinin (Sigma) medium (or plasminone). Fetal calf serum without it? overcome by using Is the result of these treatments a toxic load on cells that produce high levels of tPA? It is to reduce or decrease. Plasminogen-free serum is De
utscb and Mertz rscienceJ (1
970) Obtained by passing serum through a column of Lysine-Sepharose 4B (Pharmazoa) as described in plQ95.

Erythropoietin (ppo) gene? A clone was created from a human fetal liver library using genetic engineering techniques. Lambda-HBEPOFL13 w Naming E
The PO clone has the nucleotide 9 sequence and amino acid sequence shown in Figure aB13. Clone Lambda-HEPOF
'L+3 was inserted into the pc+toz3(B) vector and CO5-
■The cells were infected. Purified psismi) 'Each 8μ9 of DNA? Using the DEAE-dextran method:
Infect X106CO8-1 cells? C(S o m p
a yr a, c, etc., rProc, Natl, Ac
ad, Sci, J 78: 7575-7578 (1
981) and Luthman et al. “Nuc, Ac1
dsRes,, j l 1: 1295-1308 (
1983)). After 12 hours, the cells were washed, treated with chloroquine (0.1mM) for 72 hours at 37°C, washed again, and treated with 10% fetal bovine serum 10%? 10 m of culture medium for 24 hours. Change this medium to 74 mA serum-free medium,
Collected after 48 hours.

Production of immunoactive EEPO(a)'g Sherwood
and Goldwasser (Blood, 54
: 885-893 (1979)) and 300 n!
It turns out that there is /MAT.

Lambda-HEPOF'L 13 containing p91023 (B
) The vector was also infected with C08-1 cells without chloroquine treatment and grown in the same manner as described above. In vitro biological activity of EPO 1=-j, by colony formation assay with mouse fetal liver cells, the source of CFU-E;
Measured by H-thymidine uptake assay using lung cells from mice injected with phenylhydrazine]7.
Anl (T) values of 2 U/m and 3 U were obtained, respectively.

In addition, the in vivo biological and physical characteristics of EPO are shown in hypoxic mice and fasted rats. + (cFU-1i1: ) x and 2, respectively.
(3H-Tl)y) U/me q) It's worth it.

(Creation of vector PK l-4) 71 sva adjacent to the dihuman 90 vacate reductase.

Plasmid psV2DHPR containing the early region promoter, SV40 enzyme, small antigenic intron and SV40 sF rearenyl sequence (Subramani et al., Mo1Cell Biol J I: 854
BamH knee PvuPvu from -864 (1981)
II Ment (Fragment A) 1.7. The remaining fragment is vector p91023 as follows:
Obtained from (A). I) 91023 (A) yx Mono-Pst near the adenoviral promoter, linearized the plasmid by digesting with PstI in the cytoplasm, and then ligated to the synthetic Pst, I-EcoRI converter.

Restart yJ (PstI on redundant PstI site: Eco
Make RI 9-ite resistance: 91023 (B')) or
Or treated with DNA polymerase ■ dog fragment and Pst Escite? Delete and synthesize EcoRI linker (
/i: Binding and restarting (Eco to the original PstI site)
RI site? Make=91023(B)). p910
23 (Fragment A and fragment p91 derived from Bl
023('') to create two new plasmids.These plasmids)'ll1EcoRnieP
Bring either the Pst site ρ1 or the PstI-EcoR site to the original PSt site. P.S.
Plasmid F containing the Pstney EcoR site where the site is closest to the adenovirus main late promoter;
-pc+1oz3(c).

Vector p91023(c)'r XboI T: Complete VC degradation1. .

The resulting linearized DNA 1i with cohesive ends was blunted in an end-filling reaction with the E. coli large fragment of DNA polymerase. 340 bp Hina including SV40 enhancer made as follows
II knee EcoRI fragment? The above DNA Ic
Combined.

SV40 power including the SV40 origin of replication Hln
dIII-PvuII fragment resistant plasmid 9π
Insert it into the lac (Little etc., Mal Bio
1 Med, 1: 473-488 (1983)).

This πlac vector was transformed into π1acDNA by &mH engineering.
y decomposes, and large fragments of DNA polymerase produce sticky ends? Fill in this DNA y Hj ndI
It was prepared by decomposition with II.

The obtained cilasmid (π5VHP1ac) was PvuI
Is BamH nibbuty capable of binding to the I blunt end?
It went up. EcoRI-IHinujI■I fragmenton 5VHPIF]e and replicative plasmid bioorigin? E of PSVOd (Mellon et al. above) including
cm RI-Hi n d ee fragment, and the ieta et al. 1.71 niblasmid” pSVHPod
Missing 11 choices and 1 choice. SV40 origin/enhancer? 340 bp EcoR knee Hi of PSVHPod containing
Making ndI fragment, DNA polymerase■
The large fragment was made blunt at both ends. Hind
III-gcmRI flag, the rat orientation is such that the BamHI ``j'' in that fragment is closest to the VA gene (p91023(c
o/Xho/Mo smoothing ÷ Ec oR engineering/HinaIII order smoothing SV4 Q origin + enhancer) ke pES l
It was named 05. Plasmid pas l O5'rB
digested with amHI and PvuII, and with PVuE alone, and with Ba containing the adenovirus main late promoter.
mHney PvuII, ylagly) (fragmen) B), and resistance genes (tetraticline resistance)
What about plasmids and other DNA sequences? have P
The Yu Engineering I fragment (Fragment C) was produced by Kaoru.

Fragments A, B and C were ligated and the resulting plasmid shown in Figure 14 was labeled RKI-4. Deposited as Bill's ATCCK ATCC439940.

EPO expression clone lambda HEPOF'L13) et al.
The EcoR nib IC of the small R fragment plasmid RKI-4 containing the gPo gene was degraded by COR engineering.
Tebuku rJ-7L, 7':. Koo DNA (R
KFLr3) is used to directly infect DHFR-deficient CHO, n++ cells (without degradation);
Selected and expanded as shown below. 21-1 After a total of 1 no Sokyu αθ, cells that had incorporated at least %x DHFR genes were nucleonodes”? not containing io% dialyzed Shoji bovine serum?
19ζ selection was carried out in supplemented alpha medium. After two weeks of growth in selective media.

Each pool 10-1 except for Colony Kaoru's prehet power)
Pool and replate into groups of 00 colonies,
The entire surface was grown in alpha medium containing no nucleosides. M)) Medium supernatants from pools grown prior to rexate selection were assayed for EPOVc by 7R engineering. I' Custom-made 5 EPO cows? The indicated pool was immediately subcloned and tested again, and those that were historically positive were subjected to stepwise selection (see below!t6).

With respect to pools that are r/^ according to RIA, MeIJ
mltbotrexate) (mltbotrexate, 0.
A melitrexate-resistant cow colony obtained from a culture of T1 and the corresponding culture R
The pool was assayed for EPO by IA. If one is positive, subclone this culture and also test for methotrexate concentration? increased and multiplied.

Stepwise methotrexate (MTX) selection? , cells in the presence of increased concentrations of MTX? This was done by repeated cycles of culturing. At each stage, EPOy, = was determined on the culture supernatant by RIA and by in vitro biological activity. MTX used to increase concentration at each stage is 0
．． 2 μM, 0.1 bM and 0.5 μM.

RKF'L13 DNA was inserted into microinfection 2 cells and rho cells. Obtained F'PO
The degree of expression is shown in Table 6 +IC.

Table 6 Colony Boole AR Engineering A, 3 n! 7/m/
, 42 n9/rnl (pool) 150n&/7'
L/(clone) 3H-ThY', 1.5 U/ml single colony clone o-7. RIA 90n?
/WLe(0,2C-Z) 3H-Thy -5,9U/In9 microinjector RIA 60n19/7+Il
160 ng Al 9 pool (DEPO-1) 3H-Thy 1.8 U 1 The present invention has been described in detail, including preferred embodiments.

While these disclosures? Upon review, one skilled in the art will appreciate that modifications and improvements can be made within the scope of the invention.

[Brief explanation of the drawing]

pp, la figure n p5 Sumi)' pAaD26sVpA
(31) Structure y<described. Figure 1b illustrates the structure of plasmid YpCVSVL. Figure 1C illustrates the structure of plasmid pD20. Figure 1d illustrates the structure of plasmid t'pI)17. Figure 1e illustrates the structure of plasmid pD61. Figure 2a illustrates the structure of plasmid PpIFD-6. Figure 2b illustrates the structure of plasmid pL58. Figure 2C illustrates the structure TX of plasmid F''pQ2. Figure 2d illustrates the structure of plasmid F''pQ3. mL 3 Figure 1d: ';' y Sumito' pAdD
26sVpA (3) Calaplasmi) "Illustrated explanation of the production resistance of pTPL. Figure 4 shows plasmid p'rpr, Karaplasmi)" p9
1023 will be illustrated and explained following FIG. 3. Figure 5 is an illustrative diagram following Figure 4, and from p91023 is the creation of plasmid v 11) 91023 (B)? This is to explain. FIG. 6 is a diagrammatic explanation of p91.023-ATIJ. Rabbit, Figure 7 shows human tissue plasminogen activator (
tPA) and its non-coding flanking region. Figure 8a is a diagram showing four matching CDNA clones encoding the entity t, PA protein sequence. Figures 8h to 8d are diagrams illustrating a suitable method for obtaining replicative cDNA 7 of tPA. Figures 9a to 9b are diagrams illustrating a method for producing a tPAM transformation vector. Figure 10 is a diagram illustrating a method for creating a linked vector. Figure 11 is a diagram illustrating a method for creating a co-transformation vector containing a translation actin vector. Figure 12d is a schematic diagram of a transformation vector containing a eukaryotic enhancer. m12bIffl is a diagram of a transformation vector containing a region sensitive to a trans-acting transcriptional activator. The figure shows the EPO clone Ramgoo HEPOF'
The nucleotide sequence of L13 and the amino acid sequence resulting therefrom are shown. FIG. 14 is an illustrative diagram illustrating the transformation vector pRK 1-4'x. (One of the other five people -' Drawing purification I: (no change in content) FIG, Ia FIG, lb ar) tsu4ru x 3fpe']-" Kanmichio + KunshiFIG, Ic FIG, ld F IG, l e Atenovirus, 14-year-old R1)-72FIG,
2a Asovirus, tripartite meeting-9- FIG, 2b poゝFIG, 2c FIG, 2d p9+023 p910231AI FIG, 5 FIG, 6 TCG TCCCGCTGT GCCCAG
GAG AG, AL, l; b'LuF Asp Ile 1JaLeu Leu G1*
Leu Lys Ser Asp GACATT
GCG CTG CTG CAG CTG
AAA TCG GAT71G, 7 B FIG, 8a i-1(j, 81) FIG, 8d FIG, 9'b

Claims (1)

[Scope of Claims] 1) A vector system for introducing heterologous DNA into eukaryotic cells, the vector system comprising a product gene and an auxiliary gene. 2) The auxiliary gene comprises a gene encoding a translation activator, a trans-acting transcription activator, or a fragment of genomic DNA from the eukaryotic cell to be transformed. vector system. 3) The vector system according to claim 1, further comprising a selection gene. 4) The vector system according to claim 3, in which the product gene, selection gene and auxiliary DNA are not covalently linked. 5) The vector system according to claim 1, wherein the auxiliary DNA is not a carrier DNA. 6) The vector system according to claim 1, wherein the product gene and the selection gene are covalently linked. 7) The vector system according to claim 2, wherein the auxiliary DNA contains a gene encoding a translation activator. 8) A vector system according to claim 1, comprising a promoter located within the vector operatively containing the product gene so as to regulate the expression of the product gene. 9) The vector system according to claim 8, wherein the promoter is an adenovirus main late promoter containing a tripartite leader, and the auxiliary DNA is a gene encoding a translation activator. 10) The vector system according to claim 9, wherein the promoter is an adenovirus main late promoter containing a tripartite leader, and the gene encoding the translation activator is an adenovirus VA gene. 11) The product gene and the selection gene are linked, however, the gene encoding the translation activator under the control of the promoter is not linked to either the product gene or the selection gene. Vector system described in section. 12) A vector system according to claim 2, wherein the auxiliary DNA is a genomic DNA fragment from the eukaryotic cell to be transformed. 13) A vector system according to claim 12, consisting of a plurality of randomly different fragments. 14) Vector system according to claim 13, wherein the fragment is ligated to a vector containing the product gene and/or the selection gene. 15) Vector system according to claim 14, wherein the fragment is ligated to the 5' and 3' ends of the vector containing the product and/or selection gene. 16) The vector system according to claim 2, wherein the auxiliary DNA contains a gene encoding a trans-acting transcriptional activator. 17) Claim 7, wherein the auxiliary DNA further comprises a wild-type promoter for a trans-acting transcriptional activator.
Vector system described in section. 18) Gene is E_1A polyoma large T antigen, SV
The vector system according to claim 6, which is the _4_0 large T antigen or myc gene. 19) The vector system according to claim 18, wherein the trans-acting transcriptional activator gene is not linked to a vector containing a product gene and/or a selection gene. 20) The vector system according to claim 1, wherein the product gene encodes human tissue plasminogen activator. 21) A method consisting of introducing into a eukaryotic cell a vector system for introducing heterologous DNA into the eukaryotic cell, consisting of a product gene and an auxiliary gene, under conditions suitable for transformation. 22) Claim 2 in which the cells are myeloma cells
The method described in Section 1. 23) A transformant produced by the method of claim 21. 24) A method for producing a product, which comprises culturing the transformant according to claim 23 until the product accumulates, and collecting the product from the culture. 25) Plasmid_p91023(B). 26) The plasmid of claim 25, further comprising a product gene. 27) A eukaryotic cell transformed with the plasmid according to claim 26. 28) Claim 2, wherein the cells are CHO cells.
Cell according to item 7. 29) Plasmid_pRK_1_-_4. 30) The plasmid according to claim 29, which contains a product gene. 31) A eukaryotic cell transformed with the plasmid according to claim 30. 32) Claim 3, wherein the cells are CHO cells.
Cell according to item 1. 33) The vector system according to claim 1, which comprises an immunoglobulin enhancer. 34) A cell comprising the vector system according to claim 33. 35) Claim 3 in which the cells are myeloma cells
Cell according to item 4. 36) SV_4_0 enhancer, adenovirus major late promoter, adenovirus tripartite leader, SV
A vector system according to claim 1, consisting of a bacterial plasmid containing a _4_0 polyadenylation site and an adenovirus VA gene. 37) A vector system in which the bacterial plasmid consists of the SV_4_0 origin of replication. 38) 3' where the bacterial plasmid forms an intron
and claim 36 including a 5' splice site.
Vector system described in section. 39) The vector system of claim 36, wherein the bacterial plasmid contains a DHFR_3' untranslated sequence at the 3' position of the product gene. 40) DH in which the bacterial plasmid is capable of expressing DHFR
37. The vector system of claim 36, further comprising a FR transcription sequence. 41) The vector system of claim 39, wherein the DHFR transcription sequence comprises the SV_4_0 early promoter, the SV_4_0 early polyadenylation site and the DHFR gene. 42) subtracting eukaryotic cells that have been transformed to express a selectable phenotype and product and cultured into cell populations under conditions designed to select on the basis of phenotypic expression; A method of cloning, the method comprising: (a) removing from a population of cells one or more cells that express a product in a preferential manner relative to other cells in the population; culturing the selected cells into a later cell population under conditions designed to select for the change; (c) producing in a preferential manner compared to other cells in the later cell population; The method comprises the steps of further selecting from the subsequent cell population one or more cells expressing the product. 43) The method of claim 42, wherein the selected cells of step (a) are multiple clones, each clone being grown from a single transformed cell. 44) The method of claim 42, wherein in step (a), 2 to about 500 cell clones are selected. 45) Select multiple clones in step (a), combine these clones, grow them together in step (b), dilute the cell population in step (b), distribute into multiple containers, and predict Inoculate each vessel such that the statistical cell count is one cell per vessel, culture these vessels, and select the clones found in the vessel based on the highest product yield. A method according to claim 42. 46) The method of claim 42, wherein the product provides a selectable phenotype. 47) The method of claim 42, wherein the product does not give a selectable phenotype. 48) The method of claim 1, wherein the conditions of step (b) are designed to be selected based on high expression of a selectable phenotype. 49) The method according to claim 46, wherein the eukaryotic cell is an animal cell or a cell line thereof. 50) The method according to claim 49, wherein the animal cell is a vertebrate cell or derived from a vertebrate cell. 51) The method according to claim 49, wherein the vertebrate cell is a cell line of Chinese hamster ovary cells. 52) The method of claim 7, wherein steps (a), (b) and (c) are repeated successively. 53) The method of claim 50, wherein the cells selected from the subsequent cell population are further cultured under conditions that do not select on the basis of increased expression of a selection gene. 54) The method of claim 53, wherein steps (a), (b) and (c) are repeated after culturing under conditions that are not selective based on increased expression of the selection gene. 55) The method of claim 49, wherein the cells are transformed by co-transfection with the selection gene and the product gene. 56) The method of claim 42, wherein the phenotype is resistant to the drug or to the presence of a cell surface marker. 57) Claim 42, wherein the phenotype is the ability to metabolize nutrients that are not utilized by the cell before transformation.
The method described in section. 58) The method of claim 55, wherein the co-transfected genes are not covalently linked. 59) Claims in which the co-transfected genes are covalently linked to a vector comprising a promoter, a promoter-dependent promoter, and, downstream of the promoter in sequence, a product gene, a selection gene, and a polyadenylation site. The method according to paragraph 55. 60) The method according to claim 59, wherein the last nucleotide of the stop codon of the product gene and the first nucleotide of the start codon of the selection gene are directly linked. 61) The method of claim 55, wherein the co-transfected genes are not covalently linked. 62) The method of claim 61, wherein the product gene is present in a vector comprising a promoter recognized by the cell, a promoter, and sequentially downstream from the promoter the product gene and a polyadenylation site. 63) The method of claim 62, wherein the promoter is located at the 5' end of the promoter, between the polyadenylation site and the product gene, or at the 3' end from the polyadenylation site. 64) The method of claim 63, wherein the promoting factor is a viral promoting factor. 65) The method of claim 64, wherein the cells are normally permissive to the virus from which the promoting factor was obtained. 66) The method of claim 63, wherein the promoting factor is a non-viral, eukaryotic promoting factor. 67) The method according to claim 66, wherein the promoting factor is an immunoglobulin promoting factor and the cells are a myeloma cell line that secretes immunoglobulin. 68) The method of claim 62, wherein the vector does not contain a viral origin of replication. 69) The method of claim 64, wherein the promoting factor is obtained from simian virus 40, polyoma virus, bovine papilloma virus, adenovirus, Rous sarcoma virus or herpes ciplex virus. 70) The method of claim 62, wherein the promoting factor is present repeatedly in the vector. 71) Claim 70, wherein the facilitator repeats are serial.
The method described in section. 72) The method of claim 42, wherein the preferential manner in which the cells express the product is the expression of the product in large quantities and with high activity. 73) The method of claim 42, wherein the product is an enzyme, hormone, proenzyme, enzyme inhibitor or enzyme activator. 74) The method of claim 42, wherein the product is a glycosylated protein with disulfide bridges. 75) The method of claim 42, wherein the product is a human protein selected from thromboproteins or fibrin hydrolysates. 76) The method according to claim 75, wherein the protein is tissue plasminogen activator. 77) Claim 4, wherein the selectable phenotype is resistance to methotrexate or the ability to grow in a medium containing hypoxanthine, aminopterin and thymidine.
The method described in Section 7. 78) The method of claim 46, comprising inoculating a substantially identical host genetically deficient in the product protein with the cells selected in step (c). 79) The method according to claim 78, wherein the cells are human stem cells. 80) Assay at least a portion of the cells in the cell population for the cells that produce the highest activity and the greatest amount of product, and standardize the chromosomal DNA of those cell samples with a labeled DNA probe that can be hybridized with the product gene. 2. Observing the chromosomes of the specimen and selecting cells in which DNA encoding the product is found in relatively few chromosomal sites compared to other cells in the cell population. Fourth
The method described in Section 2. 81) The method according to claim 42, wherein cells are selected by selecting cells whose chromosomal characteristics are most similar to the chromosomal characteristics of the cell before transformation. 82) The method of claim 81, wherein said characteristic is chromosome length and wherein cells containing double microchromosomes, broken chromosomes or translocated chromosomes are not selected.