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WO2001018189A9 - Procede permettant d'exprimer des sequences exogenes dans des cellules de mammifere - Google Patents

Procede permettant d'exprimer des sequences exogenes dans des cellules de mammifere

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Publication number
WO2001018189A9
WO2001018189A9 PCT/EP2000/008563 EP0008563W WO0118189A9 WO 2001018189 A9 WO2001018189 A9 WO 2001018189A9 EP 0008563 W EP0008563 W EP 0008563W WO 0118189 A9 WO0118189 A9 WO 0118189A9
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WO
WIPO (PCT)
Prior art keywords
sequences
expressed
expression
nucleus
exogenous
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PCT/EP2000/008563
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German (de)
English (en)
Other versions
WO2001018189A3 (fr
WO2001018189A2 (fr
Inventor
Daniele Zink
Original Assignee
Daniele Zink
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 Daniele Zink filed Critical Daniele Zink
Priority to AU74158/00A priority Critical patent/AU7415800A/en
Publication of WO2001018189A2 publication Critical patent/WO2001018189A2/fr
Publication of WO2001018189A3 publication Critical patent/WO2001018189A3/fr
Publication of WO2001018189A9 publication Critical patent/WO2001018189A9/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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

  • the present invention relates to a method for the expression of exogenous
  • the invention relates in particular to a method for the expression of transgenic and proviral sequences in mammalian cells, the sequences being localized specifically in the active compartment of the nucleus of the mammalian cell.
  • the invention also relates to a
  • the invention further relates to methods for identifying regulatory or other sequences which promote stable localization or integration of sequences to be expressed in active compartments of the nucleus of a mammalian cell.
  • the invention further relates to the use of the method according to the invention in the production of transgenic animals, for the production of tissues and organs for the purpose of transplantation and the use of the method according to the invention in gene therapy.
  • the invention is based on the observation that the genomes of mammals within the cell nucleus are organized in higher-order compartments. It has now surprisingly been found that a distinction is made between transcriptionally active and inactive compartments can, and that the expression of integrated exogenous sequences is also subject to this functional organization of mammalian genomes. The previously largely unknown influence of genome architecture on the expression of genes has now been specifically investigated for the first time, and strategies for efficient long-term expression of transgenes with regard to gene therapy approaches and the design of transgenic animals have been developed based on the results.
  • DNA sequences located in the nuclear and nucleolar peripheries and minor accumulations of late replicating chromatin within the nucleus are not marked.
  • the core space occupied by the type I pattern is considered in the following as an "inner compartment".
  • the results shown in Figure 2 confirm the observation for all cell types that DNA sequences with a defined replication timing occupy specific core spaces during the S phase. In this way, higher-order core compartments are built that comprise DNA with a similar temporal replication sequence, for example the inner compartment comprises the early replicating DNA sequences.
  • Results also, such as single chromosome territories built from DNA that replicates at different times during the S phase, contribute to the higher order of the core compartments.
  • Individual territories show a polar organization with DNA replicating early during the S phase (IdU-labeled), clustered in subterritorial positions that are located within the core interior, and in later S-phase stages (CldU-labeled) replicating DNA, clustered in subterritorial Positions located in the nuclear or nucleolar peripheries.
  • the alignment of the polar territories gives rise to the higher order of the core compartments (see also Figure 1).
  • Fraction is particularly suitable as a marker in connection with the present invention. Since the sample is a specific marker for DNA in R bands, it was hybridized with replication-labeled nuclei of HeLa and SH-EP N14 cells. The hybridization signal was distributed over the entire inner compartment, but from the outer compartments and
  • the functional compartmentalization of mammalian genomes during the intephase could be characterized for the first time.
  • the data show that a specific pattern of spatial genomic compartmentalization is present during all interphase stages and is inherited clonally. Different compartments include DNA sequences that belong to different
  • FIG. 1 summarizes the correlations between the functional higher order Core genome architecture and chromosome organization during mitosis and interphase.
  • the functionally different, higher-order compartments are characterized by the alignment of polar chromosome territories with clusters of early replicating DNA, which are directed towards the core, and clusters of late replicating DNA, which are in the nuclear or nucleolar
  • Peripherals are localized, built up. This results in the following picture: Distinct bands (in the order of Mbp) that alternate on mitotic chromosomes are obtained as distinguishable domains during the interphase (also called subchromosomal foci, SF), but are reorganized within the territories.
  • the distinguishable SF clusters within a territory, thereby creating a polar organization of this structure in the order of several tens to several hundred Mbp.
  • the alignment of these polar territories creates more highly ordered functional genome compartments (of the order of magnitude of giga base pairs) within the nuclei of mammalian cells.
  • Expression of integrated exogenous sequences is influenced by the genome architecture, to analyze and to use the observations described above for the targeted expression of integrated sequences in the areas of gene therapy, design of transgenic animals and retroviral diseases and for the construction of optimized retroviral and lentiviral vectors.
  • transgenic Animals It is frequently observed in connection with transgenic animals that only a certain proportion of the transgenic animals or cells express the transgenes.
  • the causes of this serious limitation in the generation of transgenic Animals mainly lies in the stochastic inactivation of transgenes, in which the integration focus and the number of copies of the trenches play a role.
  • the main integration vector systems that have been developed for gene therapy are derived from retroviruses and, more recently, from lentiviruses.
  • stable long-term expression is a major problem even when using retroviral or lentiviral vectors.
  • Introduced genes are gradually inactivated in vivo when retroviral vectors are used. The reasons for this have not yet been clarified.
  • Compartmentalization of the genomes within the cell nuclei can be used to optimize retroviral and lentiviral vector systems for gene therapy, since the distribution of GC-rich and GC-poor sequences seems to correlate with the band structure of chromosomes and the corresponding spatial order of the genomes. Above all, the knowledge can be used to characterize vectors based on their location in the nucleus and then optimize them.
  • An object is to provide a method for long-term expression of exogenous sequences in mammalian cells that is useful in various biotechnological and biomedical fields and that overcomes the problems of the prior art that have been observed particularly in the fields of gene therapy and transgenic animal design ,
  • sequences to be expressed are localized in the active compartment of the nucleus of the mammalian cell.
  • Chromatin in compartments on the core periphery and the periphery of the nucleoli as well as smaller compartments in the core interior see also Figure 1.
  • the rest of the cell nucleus is filled by a large, coherent compartment that contains the transcriptionally competent and transcriptionally active DNA.
  • the different compartments are established immediately after mitosis and are stable throughout the interphase. Different characteristics of chromatin in the different compartments clearly show that these are built up from the R or G (and C) bands of the mitotic chromosomes.
  • the "active compartment of the nucleus of the mammalian cell" is thus the large, coherent compartment illustrated in FIG.
  • the active compartment can also be clearly identified using conventional cell biological methods and evidence identify.
  • the active compartment is characterized in particular by type I replication patterns, BrUTP staining by pulse labeling, staining with antibodies against hyperacetylated histone H4 and the presence of R band sequences, such as, for example, aluminum sequences or other SINES (short interspersed repeats or sequences) ,
  • the exogenous sequences to be expressed are transferred in combination with regulatory sequences to mammalian cells, which ensure stable localization in the active compartment of the nucleus of the mammalian cell and the associated long-term expression of the exogenous sequences to be expressed.
  • Long-term expression can be achieved not only by directly integrating the exogenous sequences into active compartments, but also by introducing exogenous sequences into inactive areas and then migrating the activated genes into the active compartment. (See, for example, Brown e ⁇ /. (1997) Cell 91: 845-854.)
  • the regulatory sequences are preferably promoter and / or enhancer sequences which are particularly preferably of viral origin.
  • the regulatory ones are particularly preferred embodiments, the regulatory ones
  • Sequences that are transferred to mammalian cells together with the exogenous sequences to be expressed from housekeeping or constitutively expressed genes.
  • the regulatory sequences preferably originate from tissue-specific or inducible genes.
  • the regulatory sequences are also preferably matrix attachment regions (MAR) and locus control regions (LCR).
  • MAR matrix attachment regions
  • LCR locus control regions
  • transfecting mammalian cells with a vector carrying sequences of housekeeping genes b) selecting stably transfected cells, and c) optionally checking the integration of vector sequences into the active compartment of the nucleus.
  • the invention relates to a method for the expression of exogenous sequences, in which the sequences are specifically integrated in the active compartment of the nucleus of the mammalian cell and the exogenous sequences to be expressed are transferred in combination with sequences to mammalian cells which have a homologous recombination with endogenous ones Sequences in the active compartment of the nucleus of the mammalian cell and the associated long-term expression of the exogenous sequences to be expressed enable.
  • sequences which enable homologous recombination with endogenous sequences in the active compartment in particular SINES and particularly preferably aluminum sequences, i.e. in particular the 300 bp consensus sequence of the Alu-DNA sequence family (see e.g. Singer (1982) Int. Rev. Cytol. 76: 67-112).
  • the method according to the invention for the targeted expression of exogenous sequences in the active compartment of the nucleus of the mammalian cell particularly preferably comprises the steps: a) transfecting mammalian cells with a vector that carries SINES, preferably aluminum sequences, b) selecting stably transfected cells, and c) optionally checking the integration of vector sequences in R bands.
  • DNA elements that can be used for the stable localization of transgenic sequences in active core compartments are e.g. Matrix attachment; the chicken lysozyme gene attachment regions, 3 kb fragment, should be mentioned here (McKnight et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6943-6947).
  • the locus control regions LCRs, locus
  • Control regions can be used, for example the ⁇ -globin LCR (Grosveld et al. (1987) Cell 51: 975; Talbot et al. (1989) Nature 338: 352), the mini-LCR (7.5 kb with four DNAase I hypersensitive areas) (Forrester et al. (1987) Nucl. Acids Res. 15: 10159-10177) or the human CD2 gene LCR (2 kb 3 'flanking region) (Festenstein et al. (1996) Science 271: 1123).
  • enhancer sequences are also suitable, for example the mouse muscle creatine kinase (MCK) enhancer (207 bp, position -1256 to -1050) (Jaynes et al. (1988) Mol. Cell. Biol. 8: 62-70 ).
  • promoters of ubiquitously and constitutively expressed genes are suitable within the scope of the method according to the invention, here the promoter of the dihydrofolate reductase (DHFR) gene (Scharfmann et al. (1991) Proc. Natl. Acad. Sei. USA 88: 4626-4630) and the promoter of the mouse phosphoglycerate kinase gene (mPgK) (Bawden et al. (1995) Transgenic Res. 4: 87-104) are mentioned as examples.
  • DHFR dihydrofolate reductase
  • mPgK mouse phosphoglycerate kinase gene
  • cell-type and tissue-specific elements are also useful in the context of the invention, to be mentioned here by way of example in connection with myoblasts of the MCK enhancers (Jaynes et al. (1988) supra) in combination with the promoter of the immediate early gene of the human cytomegalovirus (hCMV) (Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895) and in connection with mammary tissue the promoter of the mouse whey protein (Whey Acidic Protein, WAP) (Velander et al. ( 1992) Proc. Natl. Acad. Sci. USA 89: 12003-12007) and the promoter of the sheep ß-lactoglobulin gene (BLG) (Schnieke et al. (1997) Science 278: 2130-2133).
  • hCMV human cytomegalovirus
  • WAP WAP
  • BFG sheep ß-lactoglobulin gene
  • Viral elements which can be used in the context of the invention, for example, are the 5 'LTR (Long Terminal Repeat) of the Moloney Murine Leukemia Virus (MuLV) (Yao and Kurachi (1992) Proc. Natl. Acad. Sei. USA 89: 3357-3361), the 5 'LTR of the Rous Sarcoma Virus (RSV) Barr and Leiden (1991) Science 29: 1507-
  • 5 'LTR Long Terminal Repeat
  • MoLV Moloney Murine Leukemia Virus
  • RSV Rous Sarcoma Virus
  • HIV Human Immunodeficiency Virus
  • the object of the invention is also achieved by a method for the expression of exogenous sequences in mammalian cells, in which the sequences are specifically integrated into DNA of the inactive compartment of the nucleus of the mammalian cells.
  • the inactive compartment containing transcriptionally inactive and incompetent chromatin is formed from compartments at the core periphery and the periphery of the nucleoli as well as smaller compartments in the core interior, which are built up from the G and C bands of the mitotic chromosomes.
  • the inactive compartment can be identified on the basis of various features, in particular by a high proportion of G / C band sequences, BrUTP incorporation not detectable by light microscopy, low H4Ac degree and the typical type III-V replication pattern.
  • Sequences in combination with regulatory sequences are transferred to mammalian cells which ensure stable expression after integration into sequences of the inactive compartment of the nucleus of the mammalian cell and the associated long-term expression of the exogenous sequences to be expressed.
  • the regulatory sequences are in particular:
  • Promoter and / or enhancer sequences which are preferably of viral origin.
  • the regulatory sequences originate from tissue-specific or inducible genes on the one hand, or from housekeeping or constitutively expressed genes on the other. The above-mentioned are particularly suitable for expression in the inactive compartment
  • Matrix attachment regions and locus control regions are Matrix attachment regions and locus control regions.
  • the method according to the invention can be used in such a way that the exogenous sequences to be expressed are combined with sequences are transferred to mammalian cells which enable homologous recombination with endogenous sequences in the inactive compartment of the nucleus of the mammalian cell and thus reduce the risk of insertional mutagenesis, since the G / C band sequences present in inactive compartments are poor.
  • sequences which enable homologous recombination can be, for example, LINES (Long Interspersed Repeats or Sequences), for example the 6400 bp consensus sequence (or parts thereof) from the L1 DNA sequence family (Singer and Skowronski (1985) TIBS 10: 119 -122).
  • LINES Long Interspersed Repeats or Sequences
  • 6400 bp consensus sequence or parts thereof from the L1 DNA sequence family
  • satellite DNA sequences can also be used.
  • the method according to the invention of the targeted localization or integration of exogenous sequences for the purpose of long-term expression in mammalian cells is particularly useful for gene therapy.
  • the exogenous sequences are e.g. genes from viruses, archaebacteria, bacteria,
  • Fungi plants, invertebrates, vertebrates, especially mammals, which are long-term expressed by retroviral, lentiviral or other integrating vectors.
  • the method according to the invention can also be used useful for screening or characterizing proviral sequences.
  • proviruses are regulated in the context of mammalian genome architecture.
  • a better understanding of proviral integration and expression is not only interesting because of the great clinical importance of viruses such as HIV, but is also a prerequisite for the optimization of gene therapy vectors.
  • the localization in the core of stable integrated wild-type proviruses must first be analyzed with regard to the functional genome compartments.
  • the proviral gene expression can be monitored by in situ hybridization to viral mRNA. Further, e.g. using the GC-poor Mouse
  • MMTV Mammary Tumor Virus
  • the invention further relates to a method for the identification of regulatory or other (eg Alu) sequences suitable for homologous recombination with endogenous sequences, which require stable localization or integration of sequences to be expressed in active compartments of the nucleus of a mammalian cell, comprising the steps: a ) Transfer of a regulatory and / or homologous fusion
  • the cell biological methods that are available to and are familiar with the person skilled in the art are e.g. to in situ or in vivo hybridization (also amplified), binding of specific proteins to sequences of the construct which can be detected by fluorescence, luminescence or specific antibodies, specific enzyme activity, e.g. Detection of integrated sequences via specific binding GFP (green fluorescent protein) fusion proteins or GFP derivatives with other spectra, in combination with replication labeling, BrUTP pulse labeling or other methods for the detection of nascent RNA, immunostaining of entire compartments, detection of compartment-specific sequences (Alu / LINE sequences, isochoric
  • the invention further relates to a method for identifying sequences which are suitable for homologous recombination with endogenous sequences and which require stable integration of sequences to be expressed in sequences of the inactive compartment of the nucleus of a mammalian cell, comprising the steps: a) transfer of a fusion of regulatory sequences, expression after recombination with sequences inactive
  • Enable compartment and sequences suitable for homologous recombination with endogenous sequences, e.g. LINE sequences, with exogenous sequences to be expressed on mammalian eggs, and b) localization of the fusion in the cell nucleus by means of cell biological and / or cytogenetic methods.
  • endogenous sequences e.g. LINE sequences
  • exogenous sequences to be expressed on mammalian eggs e.g. LINE sequences
  • the invention also relates to a method for localizing integrated exogenous sequences, in particular integrated retroviral or lentiviral sequences, in the active compartment or in the inactive compartment of the nucleus of a mammalian cell by means of cell biological methods.
  • Another object of the invention is to demonstrate useful uses and possible uses of the methods according to the invention. These and other objects are achieved by using the method according to the invention in the production of transgenic animals.
  • the invention relates to the use of the method for producing a transgenic animal, which is characterized by the expression of a transgenic product in the milk of the animal.
  • transgenic animals for the production of a biomedical product or for the production of tissues and organs for the purposes of transplantation.
  • the method can be used for the targeted change of properties, e.g. of hair, milk composition, height or weight, transgenic animals.
  • the method according to the invention can be used particularly advantageously in gene therapy.
  • the use of the method according to the invention, in which sequences suitable for regulatory and / or homologous recombination with endogenous sequences are identified, is equally useful for the production and improvement of vectors, in particular vectors for gene therapy. The same naturally applies to the screening of transgenic animals or transgenic cells in connection with the production of transgenic animals.
  • transgenic animals are also well known to the person skilled in the art, in particular the microinjection of DNA into fertilized fertilizers Egg cells (Hammer et al. (1985) Nature 315: 680-683), the injection of genetically modified embryonic stem cells (ES cells) or cells of comparable potential into an early embryo (Ramirez-Solis and Bradley (1994) Curr. Opin. Biotechnol 5: 528-533; Sims and First (1993) Proc. Natl. Acad. Sci. USA 90: 6143 -6147), the transfer of nuclei from genetically modified somatic
  • the 4.7 kb vector pGFP-Cl (Baiker et al. (2000) Nature Cell Biology, 2: 182-184) has the ability for episomal replication due to an SV40 original.
  • a omyeomycin phosphotransferase gene enables the selection of transfected cells.
  • the SV40 Origin is removed using standard cloning techniques.
  • two copies of the 300 bp consensus sequence of the Alu DNA sequence family (Singer, 1982) and the human factor IX cDNA (Yao and Kurachi (1992) Proc. Natl. Acad. Sei. USA.
  • Clones with the corresponding integrations in R bands can be detected via in situ hybridization on metaphases and intephases with simultaneous representation of the active compartment (the techniques are shown in Fig. 3-5 for the endogenous Masp2 gene). Localization of the factor IX gene in the active compartment leads to a stable and high level of expression. Corresponding cell clones can be used for gene therapy
  • Figure 1 shows the compartmentalization of the genome in the cell nucleus (right) and during mitosis (left).
  • Fig. 2 shows the pulse labeling of nascent DNA from various cell lines and primary cells from humans, mice and hamsters during the S phase (left).
  • Image sequence from top to bottom represents the chronological course of the S phase.
  • the patterns generated in the cell nuclei show the typical nucleus localization of DNA with a certain replication time.
  • the right pictures show cell nuclei 1-5 days after the S-phase pulse marking.
  • the retention of typical nuclear pattern shows that the localization in the cell nucleus of DNA is stably maintained with a certain time of replication.
  • Figures 3-5 show an example of how a single gene sequence (here an endogenous gene) can be represented within the functional genome compartments.
  • Fig. 3 The endogenous housekeeping gene Masp2 was mapped by in situ hybridization using a 2.5 kb probe on human mitotic chromosomes (blue). The gene (red signals, arrows) is in the
  • Fig. 4 The figure shows individual light-optical sections through a cell nucleus (human neuroblastoma cell) that has a type 1 replication pattern (green, Cy3-dUTP label). At the same time, the Masp2 gene became in situ
  • Hybridization detected (red, arrows).
  • the three figures in the top row show the same core level in which the replication marker (green, right) and the in situ hybridization signals (red, center) were detected simultaneously.
  • the overlay is shown on the left.
  • the corresponding three lower images show a plane of the same cell nucleus which is 1 ⁇ m away. Only one of the two gene signals is still present in this level. It can be clearly seen that the R-band-specific Masp2 gene is located in the active inner genome compartment, which among other things. can be demonstrated by a type 1 replication pattern.
  • Fig. 5 The figure shows a light-optical section through a cell nucleus (human neuroblastoma cell) that has a type III replication pattern (green, Cy3-dUTP label).
  • the type III replication pattern stains the inactive compartments on the peripheries of the nucleoli (green "rings" within the cell nucleus) and the cell nucleus. It can clearly be seen that the Masp2 gene (shown via in situ hybridization, red, arrowheads) is not localized in the inactive compartments.
  • Fig. 6 The figure shows two cell nuclei (human lung epithelial cells) with an integrated SlV vector which expresses a GFP gene as an expression marker.
  • the images on the left show: 1. Staining of the DNA using DAPI, 2. GFP fluorescence, 3. Staining of the active compartment by immunostaining with an antibody against highly acetylated isoforms of histone H4 (H4Ac), 4.
  • SIV vector detected by in situ hybridization The enlarged image on the right shows that the transcriptionally active (GFP marker gene is expressed) SIV vector (red, arrowheads) is located in the active, inner R-band compartment (dark blue), which in this case was shown by immunostaining of H4Ac.
  • This detection of the transcription-active R-band compartment represents an alternative to the detection shown in Fig. 4 using the type 1 replication pattern.

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Abstract

La présente invention concerne un procédé permettant d'exprimer des séquences exogènes, en fonction de l'architecture fonctionnelle du génome dans le noyau de cellules de mammifère. Cette invention concerne notamment un procédé permettant d'exprimer des séquences transgéniques et provirales dans des cellules de mammifère, lesdites séquences étant localisées de façon spécifique dans le compartiment actif du noyau de la cellule de mammifère. Cette invention concerne également un procédé permettant d'exprimer des séquences exogènes dans des cellules de mammifère, selon lequel lesdites séquences sont intégrées de façon spécifique dans l'ADN du compartiment inactif du noyau des cellules de mammifère. De plus, cette invention concerne un procédé permettant d'identifier des séquences de régulation ou autres, qui favorisent une localisation ou une intégration stable de séquences devant être exprimées dans le compartiment actif du noyau d'une cellule de mammifère. Cette invention concerne en outre l'utilisation du procédé selon l'invention, afin de produire des animaux transgéniques, de produire des tissus et des organes destinés à une transplantation, ainsi que l'utilisation du procédé selon l'invention en thérapie génique.
PCT/EP2000/008563 1999-09-03 2000-09-01 Procede permettant d'exprimer des sequences exogenes dans des cellules de mammifere WO2001018189A2 (fr)

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DE19942149.8 1999-09-03
DE1999142149 DE19942149A1 (de) 1999-09-03 1999-09-03 Verfahren zur Expression von exogenen Sequenzen in Säugerzellen

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WO1992001800A1 (fr) * 1990-07-20 1992-02-06 Chiron Corporation Procede de transformation integrante de levure au moyen d'elements repetitifs disperses
CA2106260A1 (fr) * 1992-09-17 1994-03-18 Robert M. Kotin Adn du site d'integration du virus humain associe a l'adenovirus et ses utilisations
US5583009A (en) * 1992-12-08 1996-12-10 University Of Washington Method of preparing recombinant proteins in transgenic animals containing metallothionein gene elements that bestow tissue-independent copy number-dependent, position-indepedent gene expression
US6110666A (en) * 1994-06-09 2000-08-29 Medical Research Council Locus control subregions conferring integration-site independent transgene expression abstract of the disclosure
US5888774A (en) * 1994-12-19 1999-03-30 Cangene Corporation Recombinant DNA molecules and expression vectors for erythropoietin
US6025155A (en) * 1996-04-10 2000-02-15 Chromos Molecular Systems, Inc. Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes
US5989914A (en) * 1996-06-03 1999-11-23 Universite Laval Integration cassette for improvement of transgenesis in eukaryotes
CA2256484A1 (fr) * 1996-06-06 1997-12-11 Novartis Ag Vecteurs comprenant des elements sar
EP0918874B1 (fr) * 1996-08-16 2001-03-07 Medical Research Council Vecteurs d'expression episomiques autoreproducteurs conferant une expression genique specifique de tissus
GB9622084D0 (en) * 1996-10-23 1996-12-18 Isis Innovation Transinduction
US6245974B1 (en) * 1997-08-06 2001-06-12 North Carolina State University Matrix attachment regions
AU5484500A (en) * 1999-06-14 2001-01-02 North Carolina State University Method for obtaining low copy transgenes by direct dna transformation

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