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WO1998017812A1 - Transinduction - Google Patents

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Publication number
WO1998017812A1
WO1998017812A1 PCT/GB1997/002918 GB9702918W WO9817812A1 WO 1998017812 A1 WO1998017812 A1 WO 1998017812A1 GB 9702918 W GB9702918 W GB 9702918W WO 9817812 A1 WO9817812 A1 WO 9817812A1
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WIPO (PCT)
Prior art keywords
transinduction
gene
sequence
vector
target
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Application number
PCT/GB1997/002918
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English (en)
Inventor
Nicholas Jarvis Proudfoot
Hilary Louise Ashe
Original Assignee
Isis Innovation Limited
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.)
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Publication date
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Priority to AU47161/97A priority Critical patent/AU4716197A/en
Publication of WO1998017812A1 publication Critical patent/WO1998017812A1/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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • This invention relates to induction or enhancement of the expression of genes in eukaryotic target cells. More specifically, the invention relates to inducing or enhancing the expression of a target gene in a target cell by a novel process known as "transinduction", which involves transcribing in the target cell an exogenous nucleic acid related to the target gene.
  • transinduction a novel process known as "transinduction” which involves transcribing in the target cell an exogenous nucleic acid related to the target gene.
  • the invention further provides vectors useful for transinduction and target cells containing such vectors.
  • the invention has applications in gene therapy, and in biotechnology generally.
  • Gene therapy also involves the expression of exogenous genes in cells. In both cases, an exogenous gene is normally introduced into a host cell which does not contain a genomic copy of the gene, or which contains a genomic copy which is mutated so that either it is not expressed, or it is expressed but the expression product is a faulty protein. There is room for improvement in all of these techniques, particularly gene therapy which has so far met with only marginal success.
  • the human ⁇ -globin locus (depicted in Figure 1) is a gene cluster extending over 70 kb of DNA, and contains five erythroid specific genes, ⁇ -G ⁇ -A ⁇ - ⁇ - ⁇ , arranged in order of their developmental expression (reviewed by Orkin, 1995).
  • the ⁇ -globin locus also contains a locus control region (LCR), a type of potent enhancer element which is often associated with regulation of expression in gene clusters.
  • LCR locus control region
  • the ⁇ -globin LCR is characterised by four erythroid specific, developmentally stable hypersensitive sites (HS), HS1-4, situated 6 -18 kb upstream of the ⁇ -globin gene.
  • a fifth constitutive HS, HS5, located upstream of HS4 may function as an insulator.
  • ⁇ -globin gene cluster Different genes in the ⁇ -globin gene cluster are expressed during different stages of development. Adults express only the ⁇ -globin gene, in erythroid tissue. The human ⁇ -globin cluster represents one of the best characterised gene loci in eukaryotes, but the extent of transcription throughout the locus has not previously been directly investigated.
  • transinduction as observed in relation to the ⁇ -globin locus involves an association between the globin gene on the plasmid with the corresponding globin gene on the chromosome by sequence homology.
  • the plasmid is transcribed, for example with HIV- ⁇ in the presence of tat, the plasmid DNA is located in a region of the nucleus which is transcriptionally active, and so relocates the chromosomal globin locus into this active region.
  • the chromosomal LCR and intergenic promoters which would normally reside in transcriptionally inactive heterochromatin are now accessible to RNA polymerase II and are therefore transcribed.
  • transinduction is believed to be generally applicable to eukaryotic cells.
  • the invention therefore provides a method of inducing or enhancing the expression of at least one target chromosomal gene in a target cell, which method comprises:
  • the invention has several potential advantages over conventional ways of expressing a particular polypeptide in a cell type which does not normally express it.
  • the invention may provide a higher level of expression than would be possible by expressing a polypeptide from an expression vector such as a plasmid.
  • the invention is likely to be safer than using expression vectors, which can result in aberrant or inappropriate expression.
  • the invention also provides a means of expressing genes which would be too large for expression in a conventional manner. Expression induced by the method according to the invention is likely to be longer term than expression achieved by transient transfection with expression vectors such as plasmids.
  • the resident induced gene will continue to be expressed through several cell generations, while plasmids disappear as cells divide. This problem can be overcome in yeast using yeast artificial chromosomes (YACs), but
  • YACs at present have no equivalent in mammalian cells.
  • relatively short selected nucleic acid sequences are sufficient for transinduction. For example, sequences of about 500 nucleotides have been found to work. It is unlikely that the length required to work will be dependent upon the size of the target gene. Thus, a nucleic acid sequence for transinduction is likely to be more manageable than an exogenous nucleic acid required in an expression vector to express the polypeptide.
  • the major advantage of the invention is that it enables the expression of a chromosomal gene in a cell in which it is not normally expressed, or not normally expressed to a significant or sufficient degree.
  • conventional expression vector systems enable the expression of exogenous genes which either do not have a chromosomal counterpart in the cell, or which have a faulty counterpart which is not capable of being expressed or which expresses a faulty product.
  • the transinduction sequence is a nucleic acid sequence which is actively transcribed, or capable of being actively transcribed under suitable conditions, in the target cell, and which upon transcription is capable of transinducing the target gene.
  • the transinduction sequence preferably does not comprise a coding region corresponding to the full length target gene coding region. It may be preferable that while the transinduction sequence is capable of being transcribed in the target cell, it is non-coding in the sense that the transcript is not capable of being translated into a polypeptide in the host cell. Only nascent transcription of the transinduction sequence is required.
  • any undesirable side effects associated with the expression of the polypeptide may be avoided.
  • Such side effects might include for example an immune reaction against a non-native polypeptide which is encoded by a transinduction sequence comprising only a part of the coding region of a native gene.
  • Various methods will be known in the art for ensuring use of a non-coding region, for example introduction of a frameshift or other mutation, removal of the polyadenylation signal or use of a fragment not containing a polyadenylation signal.
  • the transinduction sequence may be homologous to a fragment of the target gene, or to a region of the chromosome on which the target gene is located, either upstream or downstream of the target gene but sufficiently closely located to the target gene for transinduction to occur.
  • the homology between the transinduction sequence and the cellular chromosome does not necessarily need to be 100% homology.
  • the homology may be for example around 50%, or at least 60%, or at least 70% or at least 80%, or at least 90%.
  • the degree of homology needs to be sufficient for the purposes of transinduction. A higher degree of homology may promote stronger transinduction than a lower degree of homology.
  • the transinduction sequence as already noted is preferably not a full length gene coding sequence.
  • the transinduction sequence may be as short as a few tens or a few hundreds of nucleotides in length. However, longer transinduction sequences are not excluded and a transinduction sequence may be several kilobases or several tens of kilobases long.
  • a preferred range is between 50 or 100 base pairs and 50 or 100 kilobase pairs, more preferably 500 base pairs to 5 kilobase pairs.
  • transinduction of a target gene works in the presence of an actively transcribing sequence homologous to the gene itself, or to intergenic nucleic acid or to another gene in the same region of the chromosome.
  • the distance between the target gene and the chromosomal sequence to which the transinduction sequence is homologous may be any distance within which the transinduction effect occurs. It will be simple to determine whether a particular distance is too great in relation to any particular target gene.
  • transinduction of the ⁇ -globin gene itself occurs in the presence of a transinduction sequence homologous to the LCR region, some 70 to 80 kb away from the ⁇ -globin gene.
  • Globin genes are unusually small genes, in the order of one or two or only a few kb.
  • the average length of human genes is in the region of 100 kb and some unusually large genes such as the utrophin gene are one or more megabases long. Thus, the distance over which transinduction works for different genes is expected to vary.
  • the transinduction sequence may be in the reverse orientation in the vector with respect to the promoter, compared to the chromosomal sequence to which it corresponds. Transinduction works independently of the orientation of the transinduction sequence.
  • the vector containing the transinduction sequence may be an expression vector such as a plasmid. Alternatively, it may be a viral vector. The vector employed needs to be able to enter the nucleus of the target cell, therefore in the case of a viral vector this will usually be a retroviral vector.
  • the promoter element operably linked to the transinduction sequence may be a constitutively acting promoter element, or it may be a conditional promoter element which allows transcription of the nucleic acid sequence only under certain conditions. Those conditions might include the presence of an exogenous factor such as a drug, or a cellular factor only present in certain cell types.
  • Conditional promoter elements include regulatable promoter elements which may be inducible or repressible. The purpose of the promoter element is to ensure that the transinduction sequence is capable of being transcribed in the target cell.
  • Particularly useful promoter elements for use in the invention are strong promoters such as the CMV promoter.
  • Suitable transcription factors can include transcription factors which are specific to certain genes, or which are specific to certain cell types. Examples of cell type specific transcription factors are the muscle tissue MyoD transcription factor, the liver HNF transcription factor and the NF ⁇ B transcription factor active in B and T cells. A person skilled in the field will know which transcription factors are appropriate for a particular target gene or target cell type.
  • One or more transcription factors may be provided, for example by introducing one or more expression vectors, capable of expressing the factor or factors, into the target cell. It may be convenient to employ a vector which both expresses the necessary transcription factors and contains the transinduction sequence.
  • the target gene may be a gene which is located in a gene cluster, such as the ⁇ -globin gene cluster.
  • a gene cluster is a group of related genes usually located relatively close together on a chromosome. Gene clusters generally do not contain intervening unrelated genes. Other examples of gene clusters are the major histocompatibility (MHC) cluster and immunoglobulin gene cluster.
  • the target gene may be an individual gene without closely related genes nearby.
  • MHC major histocompatibility
  • the chromosomal distribution of genes in the human genome will be better understood when the Human Genome Project reaches its conclusion. Already however there is a significant amount of information available on the structure of chromosomes and the distances between genes. The inventors have so far achieved transinduction over distances of 50 to 100 kb between the target gene and the chromosomal site homologous to the transinduction sequence. It is expected that transinduction will be possible over very much greater distances.
  • Transinduction according to the invention is not limited to target genes for which transcription is catalysed by RNA polymerase II.
  • transinduction may also be applied to target genes from which ribosomal and other cellular RNAs are transcribed, catalysed by RNA polymerase I or III. It is known that intergenic transcription occurs in the ribosomal gene locus.
  • Gene therapy applications of the invention include for example enhancement of expression of tumour suppressor genes such as p53 and RB in cancer treatment.
  • tumour suppressor genes such as p53 and RB
  • the fact that in general a gene defect will be present in all cells of the individual would at first appear to limit the possible benefit.
  • genes in the body which are normally redundant but one of which might be similar enough to the defective gene to provide a replacement for the defective gene function.
  • dystrophin gene mutations in which lead to the X-linked disease muscular dystrophy.
  • DMD Duchenne muscular dystrophy
  • utrophin an autosomally-encoded homologue
  • the utrophin gene therefore presents an ideal candidate for a target gene for transinduction. Moreover, both the dystrophin and the utrophin genes are unusually large (2.5 megabases in the case of dystrophin) so that the direct transfection of either of these genes into a target cell would be technically difficult or impossible.
  • Both dystrophin and utrophin will be separately transinduced in muscle and non-muscle cell lines. Exonic and intronic fragments from each gene will be inserted in front of a potent promoter element and the resulting constructs will be transfected into the target cells, with or without a second expression vector for muscle specific transcription factors (such as MyoD). Expression of dystrophin and utrophin in the cell lines will then be monitored by probing for mRNA or protein products (using specific antibodies). A successful outcome will lead to a direct attempt to transinduce these genes in animal models, as a form of gene therapy.
  • muscle specific transcription factors such as MyoD
  • the invention may be similarly applied to other genetic disorders, for example cystic fibrosis and haemophilia.
  • Some forms of haemophilia result from underexpression of normal genes; transinduction of e.g. factor XIII or factor IX may therefore be a viable option for gene therapy for haemophilia.
  • the invention further provides reagents for use in that method, such as vectors containing transcriptionally active nucleic acid sequences for transinduction, alone or in combination with vectors encoding suitable expression factors. Also provided by the invention are target cells containing one or more transinduction sequences.
  • HIV- ⁇ contains an Ava I - Hint I fragment of the HIV promoter joined to a Pvu II - Xba I fragment (19506 - 22709) of the ⁇ - globin gene by insertion into pUC18 polylinker between the Ace 65I (flushed) and Xba I .
  • a fragment from co-ordinates 39267 to 43759 containing the A ⁇ gene was cloned as a blunt ended fragment into the filled in Bam HI site of pUC 18 to create pUCA.
  • ASV- was then created by insertion of the SV40 enhancer into the filled in Asp 718 site of pUCA.
  • ASV- ⁇ FLANK was created by deletion of a fragment from the flanking region of ASV- corresponding to the sequences from the Dra I site (41022) to the Aat II site in pUC18.
  • GSV- was constructed by replacing A ⁇ sequences in ASV- (Bst Ell 40787 - 43759) with G ⁇ sequences (Bst Ell 35871 - 39208).
  • GSV- ⁇ FLANK was created by deletion of G ⁇ sequences from GSV- (Dra I 36106 - 39208).
  • GSV-FS and GSV- ⁇ FLANK FS were created by filling in only the Bst Ell site in the third exon (35871) of GSV-.
  • ⁇ SV- ⁇ FLANK was constructed by deletion of a fragment (Sty I 63839 - 64301) from p ⁇ E (Proudfoot et al., 1992).
  • ssDNA NRO probes were M13 clones made by insertion of the following fragments into the phage DNA polylinker sequence. The number of A residues complementary to the labelled U residues in the NRO transcripts are shown after the co-ordinates of each probe.
  • the HS5 M13 probes (L17-L24) were made using DNAs isolated from a 3.3Kb HS 5 containing fragment (Li and Stamatoyannopoulos, 1994). This was partially sequenced and divided into 8 approximately equal pieces. NRO analysis
  • NRO (nuclear run-on) analysis was performed on K562 or HEL cells which had been induced with 40 ⁇ M haemin for 24 and 12 hr respectively, and HeLa cells which had been transiently transfected (at 25% confluency) for 24 hr with 10 ⁇ g of plasmid DNA.
  • Cells were harvested by centrifugation, washed with PBS and re suspended in HLB (10 mM Tris, pH 7.5, 10 mM NaCI, 2.5 mM MgCI 2 ) + 0.5% NP40. After incubation on ice (5 min), nuclei were pelleted through a cushion of HLB + 0.5% NP40 + 10% sucrose.
  • Nuclei were washed in HLB and re- suspended in an equal volume of transcription buffer (40 mm Tris, pH 7.9, 1.6 M NaCI, 10mM MgCI 2 , 40 % glycerol). Nuclei treated with ⁇ -amanitin were pre-treated with 3 ⁇ g/ml for 5 min at 30°C. Nascent RNA chains were labelled by the addition of ATP, GTP and CTP to a final concentration of 250 ⁇ M, plus 60 ⁇ Ci [ ⁇ - 32 P-UTP] (800 Ci/mmol). Transcription reactions were carried out at 30°C for 15 min, and terminated by centrifugation for 30 sec.
  • transcription buffer 40 mm Tris, pH 7.9, 1.6 M NaCI, 10mM MgCI 2 , 40 % glycerol. Nuclei treated with ⁇ -amanitin were pre-treated with 3 ⁇ g/ml for 5 min at 30°C. Nascent RNA chains were labelled by the addition of ATP,
  • the filters were washed with 1 x SSPE/0.1 % SDS at room temperature for 30 min, and with 0.1 x SSPE/0.1 % SDS at 65°C for 20 min. Signals were quantitated using a phosphorimager. RNase protection
  • ⁇ -Globin mRNA is not induced from the HeLa cell chromosomes Transcription in the G ⁇ flanking region from the HeLa chromosomes occurs directly downstream of the poly(A) site (figure 2B). In contrast, in the other flanking regions of ⁇ , A ⁇ and ⁇ , there is a ⁇ 2 kb gap between the poly(A) site and sites of downstream transcription. This raises the possibility that transcription of the G ⁇ gene is induced in HeLa cells, and such transcription should produce stable G ⁇ -globin mRNA. To test this possibility, the G ⁇ gene bearing the frame shift mutation was transfected into HeLa cells.
  • This mutant generates the same pattern of flanking region transcription from the chromosome but allows mRNA from the transfected and chromosomal copies of the G ⁇ gene to be distinguished at the steady state level.
  • RNase protection analysis was used to differentiate between transcription of the mutant, transfected G ⁇ gene and the chromosomal G ⁇ gene, using a riboprobe which incorporates the frame shift mutation (figure 3B).
  • wild type G ⁇ was transfected to demonstrate that there is 100% cleavage at the mismatch.
  • a protected species corresponding to the transfected gene is detected demonstrating that transcription of G ⁇ is not induced at detectable levels in HeLa cells.
  • the 4 LCR HSs are transcribed (L1 , L3, L5, L7), as are the flanking regions of the ⁇ (E11 , E12), G ⁇ (G10, G11), A ⁇ (A19) and ⁇ (B11) genes.
  • a strong signal is observed over probe E3 as the DNA in this probe is present in the HIV- ⁇ clone.
  • no transcription of the ⁇ (AG1 , AG3) or ⁇ (B3) genes is detected, consistent with a lack of induction of transcription of the globin genes themselves.
  • ssDNA probes were made from a 3.3 kb fragment which contains HS5 (Li and Stamatoyannopoulos, 1994). Transcription over these probes was analysed following transient transfection of HIV- ⁇ (figure 4B). The entire HS5 fragment is transcribed indicating that the promoter lies further 5'.
  • Transfection of the A ⁇ gene demonstrates that this gene can also induce the LCR (data not shown) and intergenic transcripts (E11 - E14, B11 , B12, A19) (figure 6C). Transcription over probes G'10 and G'11 which lie upstream of G ⁇ are transcribed, whereas G'12 immediately downstream lacks a signal. This transcription pattern over G'10 to G'12 is also observed in HEL cells (data not shown) and may reflect another transcription termination event. As before, transcription of A ⁇ does not induce transcription of ⁇ (E3, E4) or ⁇ (B3). Similarly, transfection of the ⁇ gene induces the intergenic but not genic transcripts (data not shown).
  • Plasmids containing either the erythroid transcription factor (TF) GATA1 or NFE2 were obtained from Dr. Stuart Orkin of Harvard University Medical School (refs. GATA1 Nature Vol.339 page 446 1989; NFE2 Mol. Cel. Biol. Vol. 15 page 4640,1995). 2-25 ⁇ g of each plasmid were used per I50mm plate for transfections. Cytoplasmic RNA was purified following 2 day transfections and subjected to RNase protection RNA mapping using the ⁇ -globin 5' end riboprobe as shown in Figure 6A. RNA mapping procedures are all standard and are described or referred to above. Figure 6B shows a radioautograph of one such experiment.
  • TF erythroid transcription factor
  • the presence of the ⁇ -globin mRNA 5' end signal is clearly visible in the last lane next to the DNA size markers.
  • the level of induced ⁇ -globin mRNA is at about a 1% (but nevertheless significant) level as compared to the level of a typical transfection of plasmid containing the ⁇ -globin gene transcribed in the sense direction.
  • the transinduced ⁇ -globin mRNA is only detectable when the reverse ⁇ -globin and TFs are cotransfected.
  • transinducing vectors can be constructed as follows. Genic or intergenic fragments, preferably between 500 and 5,000bp are placed in the polylinker of an expression vector such as the standard bacterial plasmid pUCI9/18, adjacent to an efficient promoter for RNA polymerase II . Two promoters have been described above: 1) ⁇ , ⁇ or ⁇ -globin gene promoters linked to the SV40 enhancer
  • a vector for transinduction of the human BRCA1 gene may contain a non-coding fragment of the BRCA1 gene operable linked to the CMV promoter plus optionally a linked enhancer
  • Figure 1 Summary of the transcripts induced from the chromosome in non-erythroid cells.
  • Figure 2 Induction of transcription from the HeLa cell chromosome.
  • FIG. 1 This figure shows NRO analysis of HeLa cells transiently transfected with the plasmid GSV- ⁇ FLANK FS, which contains a frame shift mutation in the third exon of the G ⁇ gene.
  • B RNase protection analysis of G ⁇ transcription in HeLa cells.
  • the riboprobe incorporates the frame shift mutation tested in (A), and the position of the riboprobe relative to the G ⁇ gene is indicated.
  • the sizes of the probe fragments protected by the wild type and mutant G ⁇ mRNAs are indicated below the diagram.
  • the protected fragments obtained from RNase protection analysis of cytoplasmic RNA isolated from HeLa cells transiently transfected with GSV-FS and GSV- are shown.
  • HeLa cells transiently transfected with the ⁇ -globin gene fused to the HIV promoter A diagram of this construct is shown, with the HIV promoter drawn as a shaded box.
  • the HIV- ⁇ plasmid was transfected alone (- tat) or with a tat producing plasmid (+ tat).
  • the positions of the probes used relative to the e, g and b genes are shown in previous figures.
  • FIG. 5 Genic transcriptional induction (A) Diagram showing the HIV-HS2 transinducing plasmid used to transfect HeLa cells together with the tat expressing plasmid. Arrow indicated transcription start site on the HIV promoter that reads into the LCR sequence (from co-ordinates 8222-9253).
  • the co-ordinates of the probes used are shown above.
  • the Phoshoimage presented has the positions of the different regions in the human ⁇ -globin gene locus indicated.

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Abstract

L'invention concerne la transinduction qui est un système destiné à réguler l'expression d'un locus du gène par activation transcriptionnelle reposant sur un transcrit. L'invention prévoit un procédé permettant de provoquer ou de renforcer l'expression d'au moins un gène chromosomique cible dans une cellule cible, consistant à: (a) prévoir un vecteur contenant une séquence de transinduction capable de causer une transinduction du gène cible, ladite séquence de transinduction étant liée fonctionnellement à un promoteur; (b) introduire le vecteur dans la cellule cible; (c) mettre la cellule cible dans des conditions où la séquence de transinduction est transcrite et où le gène chromosomique cible est exprimé par transinduction. L'invention rend possible l'expression d'un gène chromosomique dans une cellule dont l'expression ne s'effectue pas normalement, ou dont l'expression ne s'effectue pas normalement à un niveau important.
PCT/GB1997/002918 1996-10-23 1997-10-22 Transinduction WO1998017812A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19942149A1 (de) * 1999-09-03 2001-03-15 Daniele Zink Verfahren zur Expression von exogenen Sequenzen in Säugerzellen

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991014436A1 (fr) * 1990-03-21 1991-10-03 Isis Pharmaceuticals, Inc. Reactifs et procedes de modulation de l'expression de genes par homotypie d'arn
EP0455424A2 (fr) * 1990-05-02 1991-11-06 Merck & Co. Inc. Système de cascade avec un promoteur de mammifère induit
WO1995033841A1 (fr) * 1994-06-09 1995-12-14 Medical Research Council Sous-regions de regulation du locus pour l'expression transgenique independante du site d'integration

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991014436A1 (fr) * 1990-03-21 1991-10-03 Isis Pharmaceuticals, Inc. Reactifs et procedes de modulation de l'expression de genes par homotypie d'arn
EP0455424A2 (fr) * 1990-05-02 1991-11-06 Merck & Co. Inc. Système de cascade avec un promoteur de mammifère induit
WO1995033841A1 (fr) * 1994-06-09 1995-12-14 Medical Research Council Sous-regions de regulation du locus pour l'expression transgenique independante du site d'integration

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOOD W.G.: "The complexities of beta-globin gene regulation", TREND IS GENETICS, vol. 12, no. 6, June 1996 (1996-06-01), pages 204 - 206, XP002052567 *
JIMENEZ G. ET AL.: "Activation of the beta-globin locus control region precedes commitment to the erythroid lineage", PROC. NATL. ACAD. SCI. USA, vol. 89, November 1992 (1992-11-01), pages 10618 - 10622, XP002052568 *
WIELAND S. ET AL.: "Genetic and biochemical analysis of a steroid receptor", 1990, KLUWER ACADEMIC PUBL., M.N. ALEXIS & C.E. SEKERIS (EDS.), XP002052964 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19942149A1 (de) * 1999-09-03 2001-03-15 Daniele Zink Verfahren zur Expression von exogenen Sequenzen in Säugerzellen

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