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WO1997008307A1 - Systeme de production de proteines heterologues au moyen de cellules aviaires - Google Patents

Systeme de production de proteines heterologues au moyen de cellules aviaires Download PDF

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Publication number
WO1997008307A1
WO1997008307A1 PCT/KR1996/000145 KR9600145W WO9708307A1 WO 1997008307 A1 WO1997008307 A1 WO 1997008307A1 KR 9600145 W KR9600145 W KR 9600145W WO 9708307 A1 WO9708307 A1 WO 9708307A1
Authority
WO
WIPO (PCT)
Prior art keywords
epo
cells
dna
group
vector
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.)
Ceased
Application number
PCT/KR1996/000145
Other languages
English (en)
Inventor
Sun Young Kim
Kee Won Kim
Tae Han Kim
Jeong Ho Hwang
Seon Hee Kim
Sun Young Lee
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.)
IL Dong Pharmaceutical Co Ltd
Original Assignee
IL Dong Pharmaceutical Co Ltd
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 IL Dong Pharmaceutical Co Ltd filed Critical IL Dong Pharmaceutical Co Ltd
Priority to EP96927924A priority Critical patent/EP0851917A1/fr
Priority to AU67563/96A priority patent/AU6756396A/en
Priority to JP9510142A priority patent/JPH11502719A/ja
Publication of WO1997008307A1 publication Critical patent/WO1997008307A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

  • the avian system has been used for the study of gene expression in higher eukaryote for a long time.
  • One of the first viruses to be linked to tumors was the Rous sarcoma virus of chicken, and this virus was instrumental in demonstrating that the retroviral oncogene can originate from a cellular gene, leading to the concept of the protooncogen.
  • Studies of gene expression have also been done using the RSV LTR promoter, which has often be used for high level expression of heterologous genes in mammalian cells.
  • avian embryo cells have been used extensively in studies of various animal viruses.
  • the vector has a promoter selected from the group consisting of SV early promoter, HCMV MIEP and RSV LTR.
  • Fig. 1 shows the expression of the bacterial CAT gene in avian ceils.
  • DE and CEF cells were transfected with pRc/CMV containing (+) or lacking (-) the CAT sequence.
  • CAT activity was measured by determining the amount of acetylated chloramphenicol (AC) produced from 14 C-chloramphenicol. The values shown are from one representative of more than five independent assays. For this particular experiment, 10 ⁇ g of protein was reacted with 14 C- chloramphenicol for 20 min at 37 °C.
  • Fig. 2 shows the comparison of CAT gene expression between various cell types and between different promoters.
  • the three promoter-CAT fusion constructs were transfected into DE, CEF, CHO- K1 , and HeLa cells, and CAT activity was measured as described in Fig. 1.
  • S SV40 early promoter
  • C HCMV MIEP
  • R RSV LTR.
  • the values shown are from one representative of three independent assays. For this particular experiment, 10 ⁇ g of protein was reacted with 14 C-chloramphenicol for 30 min at 37 °C .
  • Fig. 6 is various EPO amino acid sequences.
  • SY, SH, HE and JM are the EPO amino acid sequences cloned by the present invention
  • AM and Gl are the EPO amino acid sequences which have been already reported.
  • the abbreviation of the amino acids are as follows: A: alanine R: arginine N: asparagine D: aspartic acid
  • the two types of BamHI cassettes which could express the gene for human glutamine synthetase was made.
  • the GS cDNA sequence was flanked by the poly A sequence from the bovine growth hormone gene and one of the two promoters, the partial MMTV LTR (from -220 to +15 from the RNA start site) or the 220 bp HSV tk promoter.
  • the BamHI fragment expressing GS was inserted into the BamHI site of pCI-neo (Promega, Madison, WI, USA), resulting in a series of pIGA.
  • the Hindlll fragment of the SY-EPO cDNA sequence was cloned into the Smal site of pIGA, generating the EPO expression vector, pIGA-EPO.
  • Fig. 9 shows the production of EPO by QT-N4D4.
  • QT-N4D4 cells were grown to confluence in a 10 cm culture dish (day 0) in M-199 containing 10 % FBS and 1 mM MSX. On day 3, the EPO level was measured. The cells were then split into 1 :3 and seeded onto 10 cm dishes. On day 6, the cells were again reached confluence, and the medium was replaced with 10 ml fresh medium containing 2 % (•) or 10 % (O) FBS. EPO levels were determined by ELISA (R & D system, Minnesota, USA)
  • Fig. 10 shows the comparison of EPO concentration in DE (•) and QT-N4D4 (O) measured by ELISA and by in vitro bioassay.
  • pSV-gEPO was derived by replacing the CAT sequence of pSV918 with the genomic EPO sequence.
  • pIGA-EPO has cDNA of EPO controlled by HCMV MIEP and the genes of NEO and glutamine synthetase (hereafter "GS").
  • GS glutamine synthetase
  • the staining solution [PBS containing 4 mM K 3 Fe(CN) 6 , 4 mM K 4 Fe(CN) 6 .3H 0, 2 mM MgCI 2 , and 400 ⁇ g per ml X-gal (in dimethylformamide)] was added to fixed cells, and incubated at 37 °C for 4 hours overnight. When the reaction was completed, cells were washed once with PBS. Stained cells were kept in PBS.
  • CAT assay was carried out as follows:
  • transfection with pCMV-CAT resulted in readily detectable levels of CAT activity in both cells.
  • the level of CAT activity was always higher in DE cells than in CEF cells.
  • the magnitude of difference in the level of CAT activity between the two cells ranged from 10- to 50-fold, depending on the experiment. This result indicated that avian cells were readily transfected with DNA and the heterologous genes could be efficiently expressed.
  • CAT activity was readily detectable in the avian cells (Fig. 2), except for the SV40 promoter in CEF cells. It indicated that the expression in avian cells are more effective than that in mammalian ceils.
  • EPO expression vectors were transfected into various cells including DE, CEF, CHO, HeLa, VERO, and 293T.
  • VERO cells because they are often used for heterologous gene expression
  • 293T cells which drive very high levels of gene expression, presumably due to both the high frequency of DNA transfection and the presence of potent viral transactivators such as EIA, EIB, and large T antigen.
  • EIA, EIB, and large T antigen Two to three days after transfection, levels of EPO in the culture supernatants were measured by the enzyme linked immunoadsorbent assay, and transfection efficiencies were determined by staining cells adhered on the culture with X-gal. Transfection efficiency was carried out by transfection of a lacZ expression vector together with an EPO expression vector as described in the section II.
  • Table 2 One representative result of this analysis is summarized in Table 2.
  • the HCMV MIEP to drive expression ofthe heterologous gene as it had already been shown to be one of the strongest promoters in avian cells as well as mammalian cells.
  • the human glutamine synthetase (GS) gene was used for amplification of the target gene.
  • the gene of interest is amplified to augment the yield of protein by using certain selectable markers in the presence of specific chemicals.
  • One of the best examples is the dihydrofolate reductase (DHFR) gene. It has been shown that the copy number of the heterologous gene and the level of respective protein increase as the concentration of methotrexate (MTX) in the medium is slowly increased.
  • MTX methotrexate

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Système d'expression de protéines hétérologues comprenant un ADN de gène hétérologue, tel qu'un ADN génomique d'érythropoïétine (EPO), un vecteur recevant l'ADN et une cellule aviaire, telle qu'une lignée cellulaire d'embryon de canard ou de fibrosarcome de caille, exprimant le gène dans le vecteur. On peut utiliser ce système afin de produire efficacement des protéines hétérologues, telles que EPO.
PCT/KR1996/000145 1995-08-24 1996-08-23 Systeme de production de proteines heterologues au moyen de cellules aviaires Ceased WO1997008307A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP96927924A EP0851917A1 (fr) 1995-08-24 1996-08-23 Systeme de production de proteines heterologues au moyen de cellules aviaires
AU67563/96A AU6756396A (en) 1995-08-24 1996-08-23 Heterologous protein production system using avian cells
JP9510142A JPH11502719A (ja) 1995-08-24 1996-08-23 鳥類細胞を用いた異種蛋白質の生産システム

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1995/26391 1995-08-24
KR1019950026391A KR970010968A (ko) 1995-08-24 1995-08-24 오리 배 세포를 이용한 에리스로포이틴의 발현 시스템

Publications (1)

Publication Number Publication Date
WO1997008307A1 true WO1997008307A1 (fr) 1997-03-06

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ID=19424385

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PCT/KR1996/000145 Ceased WO1997008307A1 (fr) 1995-08-24 1996-08-23 Systeme de production de proteines heterologues au moyen de cellules aviaires

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EP (1) EP0851917A1 (fr)
JP (1) JPH11502719A (fr)
KR (2) KR970010968A (fr)
AU (1) AU6756396A (fr)
WO (1) WO1997008307A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010505A3 (fr) * 1997-08-22 1999-05-20 Univ Guelph Production de proteines dans les oeufs
WO2003054172A3 (fr) * 2002-01-17 2004-02-19 Lonza Biologics Plc Cellules humaines auxotrophes pour la glutamine capables de produire des proteines et de se developper dans un milieu exempt de glutamine
US6861572B1 (en) 1997-11-14 2005-03-01 Origen Therapeutics, Inc. Production of proteins in eggs
GB2408980A (en) * 2003-12-09 2005-06-15 Nat Biolog Standards Board Genetic reference standard
EP1552298A4 (fr) * 2002-07-01 2006-11-08 Kenneth S Warren Inst Inc Cytokines protectrices des tissus recombinees et acides nucleiques codants associes pour la protection, la restauration et l'amelioration de cellules, de tissus et d'organes sensibles
EP1939281A1 (fr) 2003-11-03 2008-07-02 ProBioGen AG Lignées de cellules aviaires immortalisées pour la production de virus
WO2012095514A1 (fr) 2011-01-14 2012-07-19 Vivalis Système de production de protéines recombinantes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003877A1 (fr) * 1987-10-21 1989-05-05 Institut National De La Recherche Agronomique (Inr Vecteurs viraux d'integration et d'expression

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ210501A (en) * 1983-12-13 1991-08-27 Kirin Amgen Inc Erythropoietin produced by procaryotic or eucaryotic expression of an exogenous dna sequence
DE69329202T2 (de) * 1992-10-02 2001-03-29 Research Corp. Technologies, Inc. Methoden der erhöhung der sekretion von überexpremierten proteinen

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003877A1 (fr) * 1987-10-21 1989-05-05 Institut National De La Recherche Agronomique (Inr Vecteurs viraux d'integration et d'expression

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010505A3 (fr) * 1997-08-22 1999-05-20 Univ Guelph Production de proteines dans les oeufs
AU750028B2 (en) * 1997-08-22 2002-07-11 Regents Of The University Of California, The Production of proteins in eggs
US6861572B1 (en) 1997-11-14 2005-03-01 Origen Therapeutics, Inc. Production of proteins in eggs
WO2003054172A3 (fr) * 2002-01-17 2004-02-19 Lonza Biologics Plc Cellules humaines auxotrophes pour la glutamine capables de produire des proteines et de se developper dans un milieu exempt de glutamine
EP1552298A4 (fr) * 2002-07-01 2006-11-08 Kenneth S Warren Inst Inc Cytokines protectrices des tissus recombinees et acides nucleiques codants associes pour la protection, la restauration et l'amelioration de cellules, de tissus et d'organes sensibles
EP1939281A1 (fr) 2003-11-03 2008-07-02 ProBioGen AG Lignées de cellules aviaires immortalisées pour la production de virus
EP2192173A1 (fr) 2003-11-03 2010-06-02 ProBioGen AG Lignées de cellules aviaires immortalisées pour la production de virus
US8940534B2 (en) 2003-11-03 2015-01-27 Probiogen Ag Immortalized avian cell lines for virus production
GB2408980A (en) * 2003-12-09 2005-06-15 Nat Biolog Standards Board Genetic reference standard
GB2408980B (en) * 2003-12-09 2006-06-07 Nat Biolog Standards Board Genetic reference materials
WO2012095514A1 (fr) 2011-01-14 2012-07-19 Vivalis Système de production de protéines recombinantes

Also Published As

Publication number Publication date
AU6756396A (en) 1997-03-19
KR100274225B1 (ko) 2000-12-15
EP0851917A1 (fr) 1998-07-08
KR19990044135A (ko) 1999-06-25
JPH11502719A (ja) 1999-03-09
KR970010968A (ko) 1997-03-27

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