EP0055742A4 - Systeme d'amplification des genes eucaryotiques. - Google Patents

Systeme d'amplification des genes eucaryotiques.

Info

Publication number
EP0055742A4
EP0055742A4 EP19810901998 EP81901998A EP0055742A4 EP 0055742 A4 EP0055742 A4 EP 0055742A4 EP 19810901998 EP19810901998 EP 19810901998 EP 81901998 A EP81901998 A EP 81901998A EP 0055742 A4 EP0055742 A4 EP 0055742A4
Authority
EP
European Patent Office
Prior art keywords
genes
cells
cad
pala
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19810901998
Other languages
German (de)
English (en)
Other versions
EP0055742A1 (fr
Inventor
Geoffrey M Wahl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Salk Institute for Biological Studies
Original Assignee
Salk Institute for Biological Studies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Salk Institute for Biological Studies filed Critical Salk Institute for Biological Studies
Publication of EP0055742A1 publication Critical patent/EP0055742A1/fr
Publication of EP0055742A4 publication Critical patent/EP0055742A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • This invention relates generally to recombinan DNA technology and, more particularly, to improvements whereby the product of the expression of eukaryotic genes is produced in large amounts in mammalian cells .
  • each nucleotide in one strand is adjacent a complimentary nucleotide in the other strand.
  • genetic changes may be made deliberately by the introduction of a predetermined nucleotide sequence, either synthesized or isolated from one strain or species, into the genetic makeup of another strain or species.
  • the strain or species into which the recombinant sequence is introduced produces, as part of its normal processes, the protein encoded by the newly introduced DNA.
  • the modified strain or species proceeds with the normal replication process, it also duplicates the inserted sequence.
  • Various techniques are known for isolating a desired nucleotide sequence or gene from one species, or constructing that sequence synthetically.
  • Such techniques include the utilization of plasmids or phages which are broken open by restriction enzymes to allow the insertion of the isolated gene. Such plasmids or phages are then reintroduced to a suitable bacterial host species, such as E. coli, where they are capable of being replicated and wherein the protein for which they encode is expressed.
  • Another object of the invention is to provide an improved mammalian cell suitable for use in a process wherein living cells are used for replication and expression of DNA segments of interest.
  • a more general object of the invention is to provide an improved method for the production and recovery of eukaryotic gene products.
  • the method of the invention results in the production of the desired polypeptide in amplified amounts in mammalian cells.
  • Genes for the de sired polypeptide are isolated and are linked to CAD genes.
  • the genes are transfected to mammalian cells and those of the mammalian cells having functional copies of both linked genes are selected. These selected cells are further selected for resistance to substantial levels of PALA.
  • the desired polypeptide may then be recovered from the further selected PALA-resistant cells in amplified amounts .
  • the present invention takes advantage of a peculiar phenomenon respecting the resistance of mammalian tissue culture cells to PALA, a transition state analog inhibitor of aspartate transcarbamylase, one of the three enzymatic activities of the multifunctional protein CAD.
  • PALA is otherwise known as N- (phosphonacetyl) -L- aspartate.
  • CAD catalyzes the first three reactions of de novo UMP biosynthesis.
  • the phenomenon referred to is that all PALA-resistant cells have amplified amounts of the CAD gene.
  • the entire CAD gene is isolated from cells containing multicopy num bers of that gene. Preferably, such cells contain from 100-200 copies of the CAD gene. Either of three methods may be utilized for isolating the CAD gene. First, high molecular weight DNA obtained by random shearing or partial digestion with restriction endonucleases could be used.
  • phage vectors Two,3, it is possible to clone 20,000 base pairs (20kbp) of PALA-resistant syrian hamster DNA. The pieces may be then ligated to recreate a functional CAD gene in accordance with the known arrangement of the genomic DNA in syrian hamster cells.
  • a third way in which the CAD gene may be isolated is to clone a single piece of genomic DNA up to 40 kbp long using cosmid vectors.
  • Cosmid vectors are a combination of the cohesive (cos) ends of ⁇ phage and the plasmid PBR 322. These cosmids can be packaged as phage due to the locations of the cohesive ends of the vector.
  • Recombinant clones may be selected as ampicillin or tetracycline resistant bacteria because of the plasmid, which contains this marker (4,5). Functionality of the cloned gene may be ascertained by determining if transfection of CAD cells (6) produces CAD transformants .
  • the eukaryotic DNA of interest is then linked to the CAD gene.
  • the gene of interest may be, for example, a gene for a human hormone isolated from natural cell material, or may be a gene synthesized by a suitable nucleotide synthesis technique (7) .
  • a suitable host cell may be co-transfected with the CAD gene and the gene of interest in a manner which results in cells containing linked arrangements of both genes .
  • Wigler and Axel (8) have shown that co-transfection of mouse cells with the herpes TK gene and the bacterial virus ⁇ X174 results in transformants containing both DNA molecules in an apparently linked arrangement.
  • the CAD gene may be ligated to the desired gene either at convenient natural restriction sites or at synthetic restriction sites created in vitro (1) .
  • the linked DNA is then transfected in the linked form to the desired host by suitable known techniques.
  • the mammalian cells into which the linked genes have been inserted are grown and suitable techniques are utilized to select those cell cultures which exhibit the characteristics of having functional copies of both the linked genes. Such a determination may be made utilizing conventional blotting technology.
  • the resultant selected cells thereby are capable of replication of both of the linked genes and are also capable of expression of the product of those genes. A further selection is then made to select those of the previously selected mammalian cells which have a resistance to substantial levels of PALA.
  • the gene of interest will have been co-amplified along with the CAD gene in at least some of the cells. Accordingly, the product of the co-amplified gene is produced at high levels.
  • the desired polypeptide produced by the desired eukaryotic gene may then be recovered from the selected PALA-resistant cells.
  • Example I A PALA-resistant cell line with approximately 100-200 CAD genes per cell (1) is processed by known methods ' to isolate the DNA.
  • the DNA is then digested partially with endonuclease EcoR1 following standard procedures.
  • This partially digested DNA is then fractionated into fragments approximately 25-40 kbp in length using sucrose gradient centrifugation.
  • the 25-40 kbp fragments are the ligated to an appropriate EcoRl digested cosmid (e.g., MUA3 of Meyerowitz et al., 5).
  • the cosmid is then packaged in vitro into the heads of bacteriophage lambda (4) .
  • the cosmids are then used to "infect" a recA- host such a HB101.
  • Trans- formants containing the cosmids with the CAD gene inserts are located using the colony filter or plaque filter hybridization techniques (9,10).
  • Those cosmids with CAD sequences are selected and are analyzed further by restriction enzyme analysis using probes for the 5' and 3' proximal regions of a 19 kbp EcoRl CAD clone.
  • Co-transfection of mammalian cells is then effected using the CAD genes as isolated above and the gene of interest by means of the co-transfection proceedur described by Wigler and Axel (9) .
  • Conventional blotting technology is used to detect cells containing both genes and a further selection for PALA-resistant cells is made. Those PALA-resistant cells which indicate co-amplificatio of the gene of interest are then assessed by RNA (13) and DNA (14) blotting experiments.
  • Radioimmune assays, e.g. (15) to determine whether the correct protein is produce are then conducted and an estimate of the increase in the amount of expression by the desired gene may be made on the basis of the selection for different levels of PALA resistance.
  • Example II The procedures of Example I are followed except that, in order to infect the bacterial host with CAD genes for purification, a pseudophage vector is used such as de signed by Alton and Davis (16) .
  • the latter vector has the advantage of selecting against recombinants which have un dergone deletion events in the process of replication in bacterial host.
  • Example III The procedure of Example I is followed except that in isolating the CAD genes, microcells (17) are used as vehicles for introducing the large DNA molecules (i.e. multicopy CAD genes) to mammalian cells in culture.
  • Example IV The procedures of Example I are followed except that in isolating the CAD genes , synthetic lipid vesicles (18) are used as vehicles for introducing the large DNA molecules (i.e. CAD genes) to mammalian cells in culture.
  • synthetic lipid vesicles (18) are used as vehicles for introducing the large DNA molecules (i.e. CAD genes) to mammalian cells in culture.
  • Example V A procedure identical with that of Example I is followed except that mechanical microinjection is used to insert the large DNA molecules.
  • the advantages of the foregoing procedures are, basically, two-fold. First, it is possible to amplify any gene in any mammalian cell of interest using PALA-resistance as the selective marker. By using transfecting DNA obtained from cells in which it is present initially as many copies, it is unnecessary to utilize genetically marked cells, in effect, CAD minus cells.
  • the second basic advantage provided by the foregoing technique is that introduction and amplification of the desired gene in a cell which normally produces and/or exports the product of that gene will typically insure that the product is processed correctly. Thus, further processing or modification of the gene product is unnecessary in order to form the active molecule.
  • a further advantage results from the fact that the substances are produced in mammalian tissue which may provide significant advantages from the standpoint of obtaining approval for use of the substances from government agencies. It may be seen, therefore, that the invention provides an improved process for producing a desired polypeptide in amplified amounts in mammalian cells.
  • the resultant recombinant DNA containing cells produced in accordance with the invention contain multiple copies of functional gene pairs, each comprising a CAD gene linked to the DNA segment of interest.

Landscapes

  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP19810901998 1980-07-08 1981-07-07 Systeme d'amplification des genes eucaryotiques. Withdrawn EP0055742A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16684380A 1980-07-08 1980-07-08
US166843 2002-09-03

Publications (2)

Publication Number Publication Date
EP0055742A1 EP0055742A1 (fr) 1982-07-14
EP0055742A4 true EP0055742A4 (fr) 1983-06-15

Family

ID=22604906

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19810901998 Withdrawn EP0055742A4 (fr) 1980-07-08 1981-07-07 Systeme d'amplification des genes eucaryotiques.

Country Status (3)

Country Link
EP (1) EP0055742A4 (fr)
JP (1) JPS57501112A (fr)
WO (1) WO1982000158A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0103632B1 (fr) * 1982-03-15 1990-12-19 The Trustees Of Columbia University In The City Of New York Procede d'introduction de genes clones, amplifiables, dans des cellules eucaryotes et de production de produits proteiniques
US5639639A (en) * 1983-11-02 1997-06-17 Genzyme Corporation Recombinant heterodimeric human fertility hormones, and methods, cells, vectors and DNA for the production thereof
GB2153363B (en) * 1983-12-07 1988-05-05 Univ Manchester Method of preparation of cloning vector
US4663281A (en) * 1984-03-22 1987-05-05 Mass Institute Of Technology Enhanced production of proteinaceous materials in eucaryotic cells
AU582288B2 (en) * 1986-03-07 1989-03-16 Damon Biotech Inc. Vector and method for achieving high level expression in eukaryotic cells
EP0262942A1 (fr) * 1986-09-30 1988-04-06 Smithkline Beecham Corporation Transfection de cellule
EP0290144A1 (fr) * 1987-05-05 1988-11-09 City of Hope Méthode de détection de résistance naissante à des agents thérapeutiques chez des malades cancéreux
EP0319206A3 (fr) * 1987-11-30 1990-04-18 Berlex Laboratories, Inc. Amplification des gènes
US5130238A (en) * 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
WO1991014781A1 (fr) * 1990-03-19 1991-10-03 Henkel Research Corporation METHODE POUR AUGMENTER L'ACTIVITE D'OMEGA-HYDROXYLASE CHEZ LE $i(CANDIDA TROPICALIS)
AT411028B (de) * 2000-12-15 2003-09-25 Boehler Edelstahl Gmbh & Co Kg Turbinenschaufel für dampf- oder gasturbinen sowie verdichter

Also Published As

Publication number Publication date
EP0055742A1 (fr) 1982-07-14
JPS57501112A (fr) 1982-07-01
WO1982000158A1 (fr) 1982-01-21

Similar Documents

Publication Publication Date Title
US12123014B2 (en) Class II, type V CRISPR systems
WO2023046153A1 (fr) Arn circulaire et son procédé de préparation
KR101906491B1 (ko) F. novicida 유래 Cas9을 포함하는 유전체 교정용 조성물
Mulligan et al. Expression of a bacterial gene in mammalian cells
US5149636A (en) Method for introducing cloned, amplifiable genes into eucaryotic cells and for producing proteinaceous products
Climie et al. Chemical synthesis of the thymidylate synthase gene.
US5374544A (en) Mutated skeletal actin promoter
KR20220150363A (ko) 개선된 사이토신 염기 편집 시스템
EP0103632B1 (fr) Procede d'introduction de genes clones, amplifiables, dans des cellules eucaryotes et de production de produits proteiniques
EP0351585A2 (fr) Vecteur contenant le promoteur du gène codant pour la beta-actine aviaire pour l'expression du gène désiré
EP0055742A4 (fr) Systeme d'amplification des genes eucaryotiques.
CN115261363B (zh) Apobec3a的rna脱氨酶活性测定方法及rna高活性的apobec3a变体
Kishi et al. Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli.
CN107326035A (zh) 一种调控水稻粒型和叶色的去泛素化酶基因ubp5及其应用
CN113249362B (zh) 经改造的胞嘧啶碱基编辑器及其应用
Mudryj et al. Deletion analysis of the mouse alpha 1 (III) collagen promoter
EP4484558A1 (fr) Protéine cas optimisée et son utilisation
JP2001509395A (ja) 細胞又は組織特異的な人工転写調節領域の同定方法
KR20190115717A (ko) 동물 세포에서 표적 ｄｎａ의 메틸화 감소와 표적 유전자의 발현 증가를 위한 조성물, 키트, 및 이를 이용한 방법
Chang et al. Complementation of a Dictyoslelium discoideum thymidylate synthase mutation with the mouse gene provides a new selectable marker for transformation
CN115448982B (zh) TaRomo1蛋白在调控小麦雄性育性中的应用
CN113429467A (zh) Npf7.6蛋白在调控豆科植物根瘤耐氮性中的应用
US6861517B1 (en) Nucleic acid fragments, recombinant vector containing the same and method for promoting the expression of structural gene by using the same
JPH02500947A (ja) 動物細胞内でのメッセンジャーｒｎａの安定化
CN120464610A (zh) 胞嘧啶脱氨酶SvSddA、包含其的碱基编辑器及应用

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): AT CH DE FR GB LU NL SE

17P Request for examination filed

Effective date: 19820709

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19840927

RIN1 Information on inventor provided before grant (corrected)

Inventor name: WAHL, GEOFFREY M.