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WO1998010084A1 - Transactivateur de la tetracycline lie a un signal de localisation nucleaire - Google Patents

Transactivateur de la tetracycline lie a un signal de localisation nucleaire Download PDF

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Publication number
WO1998010084A1
WO1998010084A1 PCT/JP1997/003109 JP9703109W WO9810084A1 WO 1998010084 A1 WO1998010084 A1 WO 1998010084A1 JP 9703109 W JP9703109 W JP 9703109W WO 9810084 A1 WO9810084 A1 WO 9810084A1
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Prior art keywords
gene
promoter
virus
recombinant
tetracycline
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PCT/JP1997/003109
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English (en)
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Hirofumi Hamada
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Hirofumi Hamada
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Priority to AU41348/97A priority Critical patent/AU4134897A/en
Publication of WO1998010084A1 publication Critical patent/WO1998010084A1/fr

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    • 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
    • C12N15/86—Viral vectors
    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material
    • C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible

Definitions

  • This invention relates to a system for the expression of genes in animal cells. More particularly, it relates to recombinant genes which can control the expression of the introduced gene strictly, and their uses.
  • substances other than the components indispensable for the growth of cells are inductively synthesized as required. Consequently, if an exogenous gene is introduced into animal cells for the purpose of causing the cells to produce a certain protein, the protein synthesized by the constant expres- sion of the introduced gene may affect the growth of the cells into which the exogenous gene has been introduced, or may exhibit toxicity to the cells.
  • inducible promoters derived from eucaryotic cells such as the metallothionein gene (E. K. Mayo et al. , Cell, Vol. 29, pp. 99-108, 1982), heat shock genes [L. Nouer et al. , "Heat Shock Response” (edited by L. Nouer) , CRC Publishing Co.] and several hormone genes (F. Lee et al. , Nature, Vol. 294, pp. 228-232, 1981).
  • the introduced gene is slightly ex ⁇ pressed even under uninduced conditions to produce a small amount of protein.
  • tetR tetracycline repressor
  • tetO tetracvcline operator
  • tTA tetracycline-controlled tetracycline trans- activator
  • an object of the present inven ⁇ tion is to provide a gene expression system in which the expression of the introduced gene is completely or substantially suppressed under uninduced conditions to exert no influence on the growth of the cells having the gene introduced thereinto, whereas the introduced gene can be expressed under induced conditions to produce the desired protein.
  • Another object of the present invention is to provide a means for permitting the easy construction of pseudotyped retroviral vector production systems which can replace conventional packaging cell systems.
  • a recombinant gene comprising an NLS gene and the tTA gene is provided as a means for accomplishing the above objects.
  • the aforesaid NLS and tTA genes are integrated in-frame, i. e. , in such a way that the proteins obtained as their gene products exhibit the desired effects. No limitation is placed on the positions where these genes are integrated, so long as their gene products exhibit the desired effects.
  • Fig. 1 schematically illustrates, in terms of gene transcriptional units, typical recombinant genes of the present invention in comparison with well-known genes.
  • CM Cytomegalovirus promoter
  • CA Promoter comprising the cytomegalovirus enhancer joined to the ⁇ -action promoter.
  • Rx Promoter comprising the cytomegalovirus enhancer joined to the Moloney murine leukemia virus promoter.
  • Fig. 2 is a graph showing the expression efficiency in an expression system using a recombinant gene of the present invention, in comparison with the expression efficiency in the conventional gene expres ⁇ sion system developed by Bujard et al.
  • the circles represent the results obtained with CMNtTA (inventive) and the squares represent the results obtained with CMtTA (comparative).
  • the open symbols represent the expression efficiencies observed in the absence of tetracycline, and the filled symbols represent the expression efficiencies observed in the presence of tetracycline.
  • Fig. 3 is a graph showing variations in gene expression efficiency according to the type of the promoter used in a gene expression system of the present invention.
  • the circles, squares and triangles represent the expression efficiencies of the introduced gene observed when the CA, Rx and Tet promoters were used for NtTA, respectively.
  • the open symbols represent the data obtained in the absence of tetra- cycline, and the filled symbols represent the data obtained in the presence of tetracycline.
  • Fig. 4 is a graph showing variations in gene expression efficiency according to the concentration of tetracycline, for several gene expression systems of the present invention (CMNtTA, inverted triangles; CANtTA, triangles turned sideways; TetNtTA, triangles; RxNtTA, squares) and conventional systems employed as compara ⁇ tive examples (TetZ alone, diamonds; CMtTA, circles).
  • CCNtTA inverted triangles
  • CANtTA triangles turned sideways
  • TetNtTA triangles
  • RxNtTA squares
  • TetZ alone diamonds
  • CMtTA circles
  • Fig. 5 is a set of electron micrographs show- ing the morphology of cells (or organisms) having been subjected to ⁇ -Gal staining for detecting the expression of the lacZ gene in gene expression systems (or infected cells) employed as comparative examples.
  • a and b correspond to the system using TetZ alone (comparable)
  • c and d correspond to the system using CMtTA (comparative).
  • the former (a, c) shows the cells cultured in the absence of tetracycline
  • the latter (b, d) shows the cells cultured in the presence of tetracycline.
  • FIG. 6 is a set of electron micrographs show ⁇ ing the morphology of cells (or organisms) having been subjected to ⁇ -Gal staining for detecting the expression of the lacZ gene in gene expression systems (or infected cells) in accordance with the present invention.
  • e and f correspond to the CMNtTA system
  • g and h correspond to the CANtTA system
  • i and j correspond to the RxNtTA system
  • k and 1 correspond to the TetNtTA system.
  • the former e, g, i, k
  • the latter shows the cells cultured in the presence of tetracycline.
  • the present invention also provides the afore ⁇ said recombinant gene into which a promoter gene indis- pensable for the expression of those genes is also integrated. Usually, it is advisable to integrate the promoter so as to be located upstream of the aforesaid NLS and tTA genes. According to the present invention, any promoter can be used without regard to its origin and type, so long as it enables the expression of the aforesaid NLS and tTA genes.
  • the NLS gene which can conveniently be used for the purposes of the present invention is one derived from the large T pro ⁇ tein (or antigen) of SV40 virus
  • the promoters which can conveniently be used include the CM promoter of cytomegalovirus, the Rx promoter comprising the enhancer of cytomegalovirus joined to the promoter of Moloney murine leukemia virus, the CA promoter comprising the enhancer of cytomegalovirus joined to the promoter of ⁇ -actin, and the Tet promoter of the tetracycline gene, though the present invention is not limited thereto.
  • a gene encoding a foreign protein may further be integrated into the aforesaid recombinant gene.
  • the recombinant gene of the present invention is such that its expression can be strictly controlled by the presence or absence of tetra ⁇ cycline. Consequently, even when the expression of any of the aforesaid genes included in the recombinant gene exerts an adverse influence on the growth of cells containing the recombinant gene, its expression can be suppressed almost completely. Moreover, its expression can also be enhanced in some cases.
  • the foreign protein contemplated in the present invention can be any protein according to the intended purpose.
  • Specific examples of the foreign protein include, but are not limited to, viral envelope proteins, hormones, cytokines, and the receptors for hormones and cytokines.
  • the above-defined recombinant gene may conve ⁇ niently be used in the form of a plasmid constructed by carrying the recombinant gene on a specific vector.
  • the present invention also provides viruses containing the aforesaid recombinant gene.
  • the preferred virus is one using an adenovirus as the host virus.
  • the adenovirus there can conveniently be used both a mastadenovirus whose host is a mammal and an aviadeno- virus whose host is a bird, but the former is preferred.
  • the present invention also provides animal cells transfected with the aforesaid vi- ruses.
  • Preferred animal cells are mammalian cells transfected with mastadenoviruses in accordance with the present invention.
  • Specific examples of such mammalian cells include A375 cells (ATCC CRL-1619; a human mela- noma cell strain), T98 cells (ATCC CRL-1690; a human cerebral tumor cell strain), A172 cells (ATCC CRL-1620), U373 cells (ATCC HTB-17) and murine 3T3 cells (ATCC CCL-163).
  • a recombinant adenovirus is first generated by joining the tetracycline transact ivator (tTA) gene to the downstream side of the promoter of cytomegalovirus (i.e., the CM promoter; see M. Gossen et al. , Proceedings of National Academy of Science, USA, Vol. 89, pp. 5547-5551, 1992).
  • tTA tetracycline transact ivator
  • another recombinant adenovirus is generated by joining the lacZ gene, which is a reporter gene for detecting the ability to activate the expression of the tTA gene, to the downstream side of the tetracycline-controllable tetracycline promoter (i.e., the Tet promoter; see M. Gossen et al. , Proceedings of National Academy of Sci ⁇ ence, USA, Vol. 89, pp. 5547-5551, 1992). Then, gene transduction is performed by cotransfecting animal cells with these two types of adenoviruses.
  • an adenovirus (hereinafter referred to as the first virus) containing a gene into which an appropriate promoter and NtTA are integrated can be used in combination with a recombinant adenovirus (hereinafter referred to as the second virus) containing a gene into which the gag-pol gene partici ⁇ pating in the replication of retroviruses is integrated downstream of the tetracycline-controllable tetracycline promoter (i. e.
  • the Tet promoter and a recombinant adenovirus (hereinafter referred to as the third virus) containing a gene into which a gene encoding the enve ⁇ lope protein of a retrovirus is integrated downstream of the Tet promoter.
  • This combination provides different uses.
  • a high-level expression system for retroviruses can be provided by infecting animal cells with the aforesaid group of viruses.
  • a sample of particular animal cells is co- infected with the first, second and third viruses.
  • another sample thereof is co-infected with the first and second viruses. Then, the trans ⁇ duction efficiencies of the gene in both samples are detected. If the transduction efficiency of the gene in the latter sample is as high as (or slightly lower than) that in the former sample, it may be presumed that the tested cells have been infected with any retrovirus.
  • the present invention makes it possible to deter ⁇ mine whether animal cells are infected with a retro- virus.
  • the plasmid pUHD15-l (see M. Gossen et al. , Proceedings of National Academy of Science, USA, Vol. 89, pp. 5547-5551, 1992) was cleaved with Xhol and Hindlll to obtain a 2.3kb CMtTA fragment. After this fragment was made blunt-ended, it was inserted into the Swal site of the plasmid pAxcw to construct the cosmid pAxCMtTA (the restriction enzyme Swal was purchased from Boehringer Mannheim and all other restriction enzymes were purchased from New England Bio Labo unless other ⁇ wise noted). Then, according to the method of Saito et al. (S. Miyake et al.
  • human 293 cells (ATCC CRL-1573) were transfected with the above cosmid and the DNA-TPC (terminal peptide complex) of human adenovirus 5 to generate a recombinant adeno ⁇ virus (AxCMtTA) (see Fig. 1).
  • the tTA gene was excised as an about lkb fragment by cleaving pUHD15 ⁇ l with Xbal, making the resulting linear DNA blunt-ended, and then cleaving it with BamHI.
  • the plasmid pRx-nZpA was constructed by adding the synthesized XR30-PK sequence (—CC ATG GAT AAA GCT GAA TTT CTC GAA GCT CCT AAG AAG AAA CGT AAG GTA GAA GAT CCT AGG AAT TC-— ; see D. Kalderon et al. , Cell, Vol. 39, pp. 499-509, 1984) to the 5' -end of the lacZ sequence of the plasmid pRxLacZ [obtained from Dr.
  • the plasmid pRxNtTA was constructed by cleaving the plasmid pRx-nZpA with EcoRI, making the resulting linear DNA blunt-ended, and ligating the aforesaid tTA gene into the BamHI site thereof.
  • the nuclear localization signal (NLS) of XR30-PK was joined to the tTA gene so as to be in-frame with the N-terminus of the protein en ⁇ coded by the tTA gene.
  • the plasmid pTetZ was constructed by cleaving the plasmid pRx'nZ [obtained from Dr. Wakimoto (as described above)] with Xbal and BamHI to obtain an about 3. lkb NLS-lacZ fragment, and inserting this fragment into the Xbal/BamHI sites of the plasmid pUHD10-3 (see M. Gossen et al. , Proceedings of National Academy of Science, USA, Vol. 89, pp. 5547-5551, 1992).
  • This pTetZ was cleaved with Xhol and Hindlll, and then treated with T4 DNA polymerase (purchased from New England Bio Labo) to generate blunt ends.
  • the plasmid pTetNtTA was constructed by cleav ⁇ ing the plasmid pRxNtTA with Ncol and BamHI to obtain an about 1. lkb fragment, and inserting this fragment into the NcoI/BamHI sites of the plasmid pTetZ.
  • the plasmid pCMNtTA was constructed by cleaving the plasmid pUHD15 ⁇ l with EcoRI, making the resulting linear DNA blunt-ended, cleaving it with Xhol to obtain a 0.77kb CM promoter fragment, and cloning this fragment into the Xhol site of the previously constructed plasmid pTetNtTA which had been cleaved with Kpnl and made blunt-ended.
  • CMNtTA recombinant adenovirus
  • the CMNtTA gene was excised as a 2.3kb fragment by cleaving the plasmid pCMNtTA with Xhol and Hindlll, treated with T4 DNA polymerase to generate blunt ends, and then cloned into the Swal site of the cosmid pAxcw to construct the cosmid pAxCMNtTA. Thereafter, the recombinant adeno ⁇ virus AxCMNtTA was generated according to the above- described method of Saito et al. (see Fig. 1).
  • HeLa cells were infected with the generated recombinant adenoviruses AxCMtTA and AxCMNtTA in the MOI range of 10 to 1250.
  • the gene expression in the system using the NtTA-carrying adeno ⁇ virus was about 2.7 times as high as that in the system using the tTA-carrying adenovirus.
  • the plasmid pCAGGS (see H. Niwa et. al. , Gene, Vol. 108, pp. 193-200, 1991) was cleaved with Sail and Hindlll. The resulting fragment was made blunt-ended and combined with the Clal linker to construct the plasmid pCAcc. After this plasmid pCAcc was cleaved with EcoRI and made blunt-ended, an about 1.5kb fragment, obtained by cleav ⁇ ing the plasmid pTetNtTA with Kpnl and Hindlll and making the resulting DNA fragments blunt-ended was inserted thereinto.
  • an about 3.6kb fragment obtained by cleaving the plasmid pRxNtTA with Xhol and Hindlll and making the resulting DNA fragments blunt- ended was inserted into the Swal site of the plasmid pAxcw to generate a recombinant adenovirus (AxRxNtTA) (see Fig. 1).
  • An about 1. lkb fragment obtained by cleaving the plasmid pRxNtTA with Ncol and BamHI was inserted into the NcoI/BamHI sites of the plasmid pTetZ con ⁇ structed in Example 1 to generate pTetNtTA.
  • HeLa cells were infected with the generated recombinant adenoviruses AxCANtTA, AxCMNtTA, AxRxNtTA and AxTetNtTA at a MOI of 250.
  • the degree of gene expression was 85.5% in the system using the CA promoter, 82.7% in the system using the CM promoter, 82.4% in the system using the Rx promoter, and 39.9% in the system using the Tet pro ⁇ moter.
  • the gene ex- pression was completely suppressed in all of the four systems.
  • Example 1 results and the results of Example 1 are summarized in Table 1, indicating that the CA, CM and Rx promoters exhibited almost equal gene expression activi- ties, but the gene expression activity exhibited by the Tet promoter was only about a half. However, all of the systems using NtTA exhibited higher gene expression activities than the system using CMtTA (see Fig. 3).
  • HeLa cells were co-infected with the recombi ⁇ nant adenoviruses AxCMtTA or AxCMNtTA and AxTetZ gener ⁇ ated in Example 1, cultured in the presence or absence of tetracycline, and then subjected to ⁇ -Gal staining.
  • AxCMtTA and AxTetZ the recombi ⁇ nant adenoviruses
  • the plasmid pSKII+VSVG was constructed by using an about 1.6kb fragment obtained by cleaving the plasmid pLGRNL (see N. Emi et al. , Journal of Virology, Vol. 65, pp. 1202-1207, 1991) with BamHI.
  • the plasmid pTetVSVG was constructed by cleaving the plasmid pSKII+VSVG with BamHI and inserting the resulting about 1.6kb fragment into the BamHI site of the plasmid pUHD10-3. Then, according to the method of Saito et al.
  • an about 2.4kb fragment obtained by cleaving the plasmid pTetVSVG with Xhol and Hindlll and making the resulting DNA fragment blunt-ended was inserted into the Swal site of the plasmid pAxcw to generate a recombinant adenovirus (AxTetVSVG) (see Fig. 1).
  • HeLa cells were co-infected with the generated recombinant adenoviruses AxTetVSVG and AxRxNtTA, and then subjected to immunohistochemical staining with a monoclonal anti-VSVG antibody (clone P5D4, Sigma #V5507).
  • Example 5 In order to monitor the production efficiency of a retroviral vector, human cultured cells such as A375, U373, T98G and A172 were infected with the retro ⁇ viral vector MFGlacZ expressing the reporter gene lacZ (see Dranoff et al. , Proceedings of National Academy of Science, USA, Vol. 90, pp. 3539-3543, 1993).
  • the 5307bp gag-pol gene extending from nt563 (Hindlll) to nt5870 (Seal) was excised by cleaving MoMLV (see Schnich et al. , Nature, Vol. 293, pp. 543-548, 1981) with Hindlll and Seal.
  • the Tet promoter was excised by cleaving pTetZ with Xhol, making the result ⁇ ing linear DNA blunt-ended, and then cleaving it with EcoRI. Then, the plasmid pTetGP was constructed by cloning the gag-pol gene and the Tet promoter into the Spel and Bglll sites of pCAcc.
  • pTetGP was constructed by a plurality of steps in which the gag-pol of MoMLV was fragmented, these fragments were subcloned into pBluescript SKII and further sub- cloned into pCAcc to construct pCA'GP, and its CA pro ⁇ moter was replaced by the Tet promoter.
  • the cosmid pAxTetGP was constructed by cloning into the Clal site of pTetGP.
  • 293 cells were co-infected with the cosmid pAxTetGP and Ad5'DNA ⁇ TPC to generate a recom- binant adenovirus (AxTetGP) .
  • the previously prepared A375/Z, T98G/Z, A172/Z and U373/Z cells were co-infected with all or some of the generated recombinant adenoviruses AxTetVSVG, AxTetGP and AxCANtTA.
  • the culture super- natants considered to contain a retrovirus were recov- ered.
  • monitor cells e.g., NIH 3T3 cells
  • NIH 3T3 cells were infected therewith in the presence of 8 ⁇ g/ml Polybrene (Sigma), and subjected to X-Gal staining after 2 days. Then, the efficiency of infection with the retrovirus was calculated.

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Abstract

L'invention concerne des gènes de recombinaison dans lesquels sont intégrés un gène de signal de localisation nucléaire (NLS) et le gène activateur de la tétracycline (tTA). L'invention traite aussi de virus contenant ces gènes de recombinaison et de cellules animales infectées par ces virus.
PCT/JP1997/003109 1996-09-06 1997-09-04 Transactivateur de la tetracycline lie a un signal de localisation nucleaire WO1998010084A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41348/97A AU4134897A (en) 1996-09-06 1997-09-04 Tetracycline transactivator having a nuclear localization signal joined thereto

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Application Number Priority Date Filing Date Title
JP8257451A JPH1080274A (ja) 1996-09-06 1996-09-06 核移行シグナルを結合したテトラサイクリン・トランスアクチベーター
JP8/257451 1996-09-06

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WO1998010084A1 true WO1998010084A1 (fr) 1998-03-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1446479A1 (fr) * 2001-09-29 2004-08-18 YUN, Chae-Ok Adenovirus recombinant a effet therapeutique ameliore et composition pharmaceutique comprenant ledit adenovirus recombinant
US10851380B2 (en) 2012-10-23 2020-12-01 Toolgen Incorporated Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US11697823B2 (en) 2016-11-24 2023-07-11 Cambridge Enterprise Limited Controllable transcription

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029442A2 (fr) * 1993-06-14 1994-12-22 Basf Aktiengesellschaft Maitrise precise de l'expression des genes dans les cellules encaryotes au moyen de promoteurs reagissant a la tetracycline
WO1994029440A1 (fr) * 1993-06-04 1994-12-22 The Regents Of The University Of California Production, concentration et transfert efficace de vecteurs retroviraux resultant de la formation de pseudotypes par la proteine g du virus de la stomatite vesiculeuse (vsv)
WO1995019368A1 (fr) * 1994-01-14 1995-07-20 Alexion Pharmaceuticals, Inc. Particules de vecteurs retroviraux intervenant dans la transduction de cellules non proliferantes
WO1997035992A1 (fr) * 1996-03-27 1997-10-02 Vical Incorporated Systemes de tetracycline inductibles/repressibles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029440A1 (fr) * 1993-06-04 1994-12-22 The Regents Of The University Of California Production, concentration et transfert efficace de vecteurs retroviraux resultant de la formation de pseudotypes par la proteine g du virus de la stomatite vesiculeuse (vsv)
WO1994029442A2 (fr) * 1993-06-14 1994-12-22 Basf Aktiengesellschaft Maitrise precise de l'expression des genes dans les cellules encaryotes au moyen de promoteurs reagissant a la tetracycline
WO1995019368A1 (fr) * 1994-01-14 1995-07-20 Alexion Pharmaceuticals, Inc. Particules de vecteurs retroviraux intervenant dans la transduction de cellules non proliferantes
WO1997035992A1 (fr) * 1996-03-27 1997-10-02 Vical Incorporated Systemes de tetracycline inductibles/repressibles

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOSSEN M ET AL: "TIGHT CONTROL OF GENE EXPRESSION IN MAMMALIAN CELLS BY TETRACYCLINE-RESPONSIVE PROMOTERS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, no. 12, 15 June 1992 (1992-06-15), pages 5547 - 5551, XP000564458 *
SONEOKA Y ET AL: "A TRANSIENT THREE-PLASMID EXPRESSION SYSTEM FOR THE PRODUCTION OF HIGH TITER RETROVIRAL VECTORS", NUCLEIC ACIDS RESEARCH, vol. 23, no. 4, 25 February 1995 (1995-02-25), pages 628 - 633, XP000569533 *
YOSHIDA Y. ET AL.: "Highly efficient vsvg-pseudotyped retroviral packaging system through adenovirus-mediated inducible gene transduction - recombinant adenovirus-mediated vesicular-stomatitis virus-G and mouse Moloney leukemia virus gag and pol gene transfer to glioma for cancer gene therapy", CANCER GENE THERAPY, vol. 3, no. 6 Conference suppl., 14 November 1996 (1996-11-14), pages s21 - s22, XP002050699 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1446479A1 (fr) * 2001-09-29 2004-08-18 YUN, Chae-Ok Adenovirus recombinant a effet therapeutique ameliore et composition pharmaceutique comprenant ledit adenovirus recombinant
EP1446479A4 (fr) * 2001-09-29 2006-04-05 Univ Yonsei Seoul Adenovirus recombinant a effet therapeutique ameliore et composition pharmaceutique comprenant ledit adenovirus recombinant
US9175309B2 (en) 2001-09-29 2015-11-03 Industry-University Cooperation Foundation Hanyang University Recombinant adenovirus with enhanced therapeutic effect and pharmaceutical composition comprising said recombinant adenovirus
US10851380B2 (en) 2012-10-23 2020-12-01 Toolgen Incorporated Methods for cleaving a target DNA using a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein
US11697823B2 (en) 2016-11-24 2023-07-11 Cambridge Enterprise Limited Controllable transcription

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