WO2001018189A9

WO2001018189A9 - Method for expressing exogenous sequences in mammalian cells

Info

Publication number: WO2001018189A9
Application number: PCT/EP2000/008563
Authority: WO
Inventors: Daniele Zink
Original assignee: Daniele Zink
Priority date: 1999-09-03
Filing date: 2000-09-01
Publication date: 2002-09-06
Also published as: DE19942149A1; AU7415800A; WO2001018189A3; WO2001018189A2

Abstract

The invention relates to a method for expressing exogenous sequences in mammalian cells depending on the functional architecture of the genome in the nucleus of the mammalian cell. The invention specifically relates to a method for expressing transgenic and proviral sequences in mammalian cells, wherein the sequences are specifically localized in the active compartment of the nucleus of the mammalian cell. The invention further relates to a method for expressing exogenous sequences in mammalian cells, wherein the sequences are specifically integrated into the DNA of the inactive compartment of the nucleus of the mammalian cells. The invention also relates to a method for identifying regulatory sequences or other sequences that promote a stable localization or integration of sequences to be expressed in the active compartments of the nucleus of a mammalian cell. Also, the invention relates to the use of the inventive method for producing transgenic animals, for producing tissues and organs for the purpose of transplantation and to the use of the inventive method in gene therapy.

Description

Process for the expression of exogenous sequences in mammalian cells

The present invention relates to a method for the expression of exogenous

Sequences depending on the functional architecture of the genome in the nucleus of mammalian cells. 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

Process 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 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 expression of exogenous sequences, and in particular long-term expression, is a central problem in various areas of biotechnology and biomedical research. This is especially true in

Relation to gene therapy, the design of transgenic animals and retroviral diseases. 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.

Understanding the functional architecture of cell nuclei is a key problem in cell biology today. The question of how mammalian genomes are functionally organized within cell nuclei is of particular interest. Although this problem has been discussed for many years, no experimental results have become known that reliably indicate functional organization principles. It is all the more surprising that it has now been possible for the first time to show how mammalian genomes are organized in relation to important core functions. From this knowledge, a variety of useful applications in the field of gene therapy and the design of transgenic animals could be developed.

The observations described below are illustratively summarized in Figure 1. Since the influence of the functional genome organization on the expression of transgenes has now been elucidated, there is now the possibility of realizing reproducible and effective long-term expression of transgenes.

Earlier studies on mitotic chromosomes indicated a functional compartmentalization of mammalian genomes, which has a clear connection with the already known band patterns and their specific isochoric compositions. A correlation of isochors from GC poor and GC rich families with G / C and R bands could be confirmed a few years ago using in situ hybridization. Although further indications were discussed that the classic band structure, which is only observed on mitotic chromosomes, is important for the genome organization with regard to important core functions such as replication and transcription, there have been no concrete indications in connection with the substructure of chromosome territories, the core equivalents of mitotic chromosomes. Rather, different and partly opposing models were discussed, so that the meaning of the organization of mitotic chromosomes, which is reflected in the band patterns, for the functional organization of the genome was still unclear. It has now been shown for the first time that mammalian genomes are functionally organized in higher ordered core compartments. DNA sequences within each compartment replicate in specific S phase stages. Higher-order compartments that build up immediately after mitosis remain stable during all interphase stages, and the organizational principle is inherited clonally. The higher order compartments are built up by the parallel alignment of polar chromosome territories. Polar chromosome territories concentrate early replicating R band and late replicating G / C band DNA at different subterritorial positions.

Various features of the higher-order nuclear compartments, such as replication timing or the degrees of acetylation of histone H4, show that perinuclear, perinucleolar and smaller internal compartments harbor the regions of the genome that are organized into the G and C bands of mitotic chromosomes (see also figure 1). A large coherent compartment within the core contains the sequences of the R bands, as could be shown, inter alia, by in situ hybridization. Only within this compartment is the chromatin transcriptionally competent and transcriptionally active at the resolution limit of light microscopy. In order to clarify the question whether compartments comprising DNA sequences with a similar temporal replication course are actually reproducibly established after mitosis and remain stable during all subsequent interphase stages, DNA from different cell lines and

Primary cells from humans, mice and hamsters are pulse-marked during the S phase. The investigations were carried out with the following cell types: primary human diploid fibroblasts (HDFs), HeLa S6 cells, human neuroblastoma cells (SH-EP N14), CHO cells (Chinese Hamster Ovary) and C2C12 mouse myoblasts. The pulse marker was with

BrdU (bromodeoxyuridine) or Cy3-dUTP (was micro-injected). The cells were fixed 30 minutes after addition of the modified nucleotides in order to obtain typical S-phase patterns. The result is shown in Figure 2, left column. Regarding the S-phase pattern, the classification by O'Keefe et al. (1992, J.Cell Biol. 116: 1095-1110) and defined five different types of patterns which represent an ordered chronological sequence during the course of the S phase. Type I shows hundreds of small foci (approximately 300 nm in diameter) distributed over the DNA inside the nucleus. DNA sequences located in the nuclear and nucleolar peripheries and minor accumulations of late replicating chromatin within the nucleus (see types IV and V) 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. A comparison with the result of pulse labeling of cells that were not fixed immediately but only after a growth period of 1-5 days after labeling, confirmed that the core compartments comprising DNA with a similar replication timing are also stably maintained in non-S-phase cells.

For additional confirmation of the clonal inheritance of the

To obtain core compartmentalization, additional experiments with IdU (iododeoxyuridine) / CldU (chlorodeoxyuridine) double pulse labeling were carried out. The results obtained in this way also confirmed that the core compartments, which comprise DNA sequences with a similar replication timing, are set up immediately after mitosis according to the pattern present in the previous interphase. Similar compartmentalization patterns (IdU type I pattern with CldU type III-V patterns) were also observed after at least two mitoses 69 hours after the initial IdU-CldU labeling. Since individual replication-labeled chromosome territories could be observed after this period, the showed

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).

In-situ hybridization experiments in combination with Cy3-dUTP replication labeling were carried out in order to clarify whether DNA belonging to specific chromosomal bands contributes to various higher-order core compartments. As a sample for in situ hybridization the H3 isochoric fraction of human DNA (Saccone et al. (1996), Gene 174: 85-94) was used. The H3 fraction usually hybridizes with a subset of R bands on human mitotic chromosomes (Saccone et al., Supra). In the course of the investigations described here, a specific hybridization with all R bands was observed, which is why this

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

Compartments of late replication excluded. These data confirmed that the DNA in R bands forms the inner compartment. This was the first time that a clear correlation between the organization of mammalian genomes during the interphase and the band organization of mitotic chromosomes was shown.

In order to check whether transcriptionally competent chromatin is actually restricted to certain core compartments, replication-labeled nuclei were stained with antiserum R 232/8, which recognizes hyperacetylated isoforms of histone H4. A comparison of the replication labeling pattern and the nuclear distribution of hyperacetylated histone H4 isoforms showed a clear correlation. Hyperacetylated histone H4 isoforms were detected in the entire inner compartment, but could not be observed in peripheral and late-replicating compartments. Because the transcriptional competence of the genome compartments is an important feature in terms of their functional

Represents properties, the analysis was extended to four cell types from three species. For all cell types, the same correlation could be found between core compartments, which were made visible by replication labeling, and the nuclear distribution of hyperacetylated histone H4 isoforms to be watched. Hyperacetylated isoforms of histone H4 are confined to the inner compartment.

Overall, the results suggest that not only the

Transcription competence, but also the process of transcription itself is the subject of the higher-order core compartmentalization. Replication labels with FITC-dUTP in combination with pulse labeling with BrUTP (incorporated into nascent RNA) and immunostaining of incorporated BrUTP showed that only the inner compartment is transcriptionally active. No noteworthy BrUTP marking was visible within the outer compartments. It could thus be shown that the inner compartment comprising the early replicating R band sequences harbors both the transcriptionally competent and the actively transcribed chromatin (see Figure 1). The above-mentioned and other cell biological techniques are known to the person skilled in the art and can be found in the relevant literature; for example, zinc et al. (1998) Hum. Genet. 102: 241-251, for a detailed description of the experiments and results mentioned, see also Sadoni et al. (1999) J. Cell Biol. 146 (6) 1211-1226.

Thus, 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

Chromosome bands belong during mitosis. R-band sequences form a coherent compartment within the core that houses the transcriptionally competent chromatin. Detectable nascent RNA synthesis is limited to the inner compartment. Figure 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.

Now that the principles of functional architecture within the cell nuclei have been uncovered for the first time, exogenous sequences can be specifically integrated into mammalian genomes and localized in the desired functional compartments. It is now also possible for the first time to ask how

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.

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. So far, there have also been many problems in gene therapy based on a lack of knowledge about functional genome organization. So far, none of the more than 200 clinical trials with gene therapy approaches have been successful (see e.g. Cristal (1995) Science 270: 404-410; Verma and Somia (1997) Nature 389: 239-242). Integration vectors are particularly attractive in connection with the treatment of monogenic diseases, since these systems theoretically allow stable long-term expression. The main integration vector systems that have been developed for gene therapy are derived from retroviruses and, more recently, from lentiviruses. However, 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. Before that

The problem of inactivation (whether through de novo methylation or chromatin restructuring, which in both cases are only hypotheses) has not been resolved, successful gene therapy with retroviral vectors remains impossible.

Studies of retroviruses from various mammalian species, including humans, have shown that the GC content of DNA sequences at the integration site corresponds to the GC content of stably integrated proviral sequences. While these phenomena have so far not been explained, the observations described above can be made about the higher order

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 ,

One solution to this problem is to provide a method for

Expression of exogenous sequences in mammalian cells, in which the sequences to be expressed are localized in the active compartment of the nucleus of the mammalian cell.

As described above, there is transcriptionally inactive and incompetent

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. In the context of the invention, the "active compartment of the nucleus of the mammalian cell" is thus the large, coherent compartment illustrated in FIG. 1, which contains the transcriptionally competent and transcriptionally active DNA and is built up from the GC-rich R bands of the mitotic chromosomes , As described above, 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) ,

In a preferred embodiment of the method according to the invention, 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 α /. (1997) Cell 91: 845-854.)

The regulatory sequences are preferably promoter and / or enhancer sequences which are particularly preferably of viral origin. In 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. On the other hand, 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). In a further embodiment, the method according to the invention for the targeted expression of exogenous sequences in the active compartment of the nucleus of the mammalian cell comprises the steps:

a) 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.

In a further preferred embodiment, 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.

These are the 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.

Other 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 β-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). Of course, 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 ).

Furthermore, promoters of ubiquitously and constitutively expressed genes (houskeeping 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.

As mentioned above, 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).

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-

1509) and the 5 'LTR (tat-regulated) of the Human Immunodeficiency Virus (HIV) (Parolin et al. (1996) Virology 222: 415-422).

The person skilled in the art is very familiar with conventional cloning techniques and other molecular biological methods; the techniques which can be used in the context of the method according to the invention can also be found in relevant manuals, for example the manual by Sambrook et al. (1989) A Laboratory Manual, 2nd Edition, Cold Spring Harbor.

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.

In the context of this invention, 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. As already mentioned above, 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.

In a preferred embodiment of the method for the expression of exogenous sequences in the inactive compartment, those to be expressed are

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. In particular embodiments of the method, 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.

Since the risk of insertional mutagenesis when inserting exogenous sequences into endogenous sequence regions with a low gene density is significantly lower than when integrating into genetically rich regions of the genome, 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. The 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). In addition, satellite DNA sequences can also be used.

As mentioned above, 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. For this reason, 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.

Of course, the method according to the invention can also be used useful for screening or characterizing proviral sequences. As mentioned above, nothing is currently known about how 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. For this purpose, the localization in the core of stable integrated wild-type proviruses must first be analyzed with regard to the functional genome compartments. In parallel, the proviral gene expression can be monitored by in situ hybridization to viral mRNA. Further, e.g. using the GC-poor Mouse

Mammary Tumor Virus (MMTV) are investigated whether proviruses can be expressed exceptionally within the GC-poor "silenced" compartments, or whether they change compartments, ie shuttle-like "migrate" to an active compartment to be expressed. This way, not only can additional important knowledge regarding gene therapy important proviruses and retroviral vectors can be obtained, vectors useful for gene therapy can also be improved and optimized with regard to localization and associated expression optimization.

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

Recombination with endogenous sequences, suitable sequences with exogenous sequences to be expressed on mammalian cells, and b) localization of the integrated fusion in the cell nucleus by means of cell biological methods.

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

Fractions), detection of the compartments via specific fusion proteins that can be visualized in vivo. The expression of the fusion can be determined by means of expression analysis, suitable reporter genes and quantitative RT-PCR. The methods mentioned above are described, for example, in the following publications: Kurz et α /. (1996) J. Cell Biol. 135: 1195-1202, in connection with in situ hybridization; Wansink et α /. (1993) J. Cell Biol. 122: 283-293, in connection with BrUTP pulse labeling; Robinett et α /. (1996) J. Cell Biol. 135: 1685-1700, in connection with the detection of binding

Fusion proteins; Zink et α /. (1998) Hum. Genet. 102: 241-251, related to replication labeling; Dobie et α /. (1996) Proc. Natl. Acad. Be. USA 93: 6659-6664, in connection with expression analysis.

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.

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. In particular, 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. Another preferred

Use consists in the use for the production of transgenic animals for the production of a biomedical product or for the production of tissues and organs for the purposes of transplantation. Furthermore, the method can be used for the targeted change of properties, e.g. of hair, milk composition, height or weight, transgenic animals.

As already mentioned, 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.

The person skilled in the art is familiar with the relevant methods for the transfer of DNA sequences to mammalian cells. In the field of gene therapy, ex vivo or in vivo infection with recombinant retro or lentiviruses are particularly suitable (Dai et al. (1992) Proc. Natl. Acad. Sei. USA 89: 10892-10895; Kafri et al. (1997) Nat. genet. 17: 314-317; Naldini et al. (1996) Proc. Natl.

Acad. Be. USA 93: 11382-11388; Scharfmann et al. (1991) Proc. Natl. Acad. Be. USA 88: 4626-4630); Verma and Somia (1997) Nature 389: 239-242).

Methods in the production of 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

Cells in enucleated egg cells (Wilmut et al. (1997) Nature 385: 810-813; Wolf et al. (1998) J. Biotechnol. 65: 99-110) and the transfer of nuclei from cells of genetically modified cells cultured in vitro to enucleated egg cells (Schnieke et al. (1997) Science 278: 2130-2133; Wilmut et al. (1997) supra) or ex vivo transfection with other integrating vectors and subsequent ones

Transplantation of stably transfected cell clones (see also examples below).

example

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. To prevent the ability to replicate episomally, the SV40 Origin is removed using standard cloning techniques. In addition, 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. 89: 3357-3361) are cloned onto the vector control of the dihydrofolate reductase gene promoter (Scharfmann et al. (1991) Proc. Natl. Acad. Sci. USA. 88: 4626-4630), 3 'fused to the SV40 polyadenylation signal.

Primary human fibroblasts in early passages are transfected with the vector. Stably transfected cell clones are produced by selection using the usual techniques for producing stably transfected cell lines. While random integrations often take place in inactive and heterochromatic areas of the genome, the homologous recombination with the largely R-band-specific endogenous Alu sequences leads to an integration in R-bands, which build up the transcriptionally active compartment during the integration phase. Homologous recombination appears to be a relatively common occurrence in mammalian cells cultured in vitro (McCreath et al. (2000) Nature, 405: 1066-1069). Efficiency is increased by the fact that the Alu sequences are present in high copy numbers in the R bands (approx. 10 ⁶ per genome (Korenberg and Rykowski (1988) Cell. 53: 391-400)). 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

Be transplanted for purposes.

pictures

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). The

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

R band lp36 at the distal end of the short arm of chromosome 1.

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 µm 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.

Claims

Expectations

1. A method for expressing exogenous sequences in mammalian cells, characterized in that the sequences are targeted in the active compartment of the

The nucleus of the mammalian cell can be localized.

2. A method of expression according to claim 1, wherein the exogenous sequences to be expressed in combination with regulatory sequences are transferred to mammalian cells, which ensure stable localization in the active compartment of the nucleus of the mammalian cell and associated long-term expression of the exogenous sequences to be expressed.

3. A method of expression according to claim 2, wherein the regulatory sequences promoter and / or enhancer sequences or others

Include sequences with regulatory function.

4. A method of expression according to claim 2 or 3, wherein the regulatory sequences are of viral origin.

5. A method for expression according to any one of claims 2 to 4, wherein the regulatory sequences are derived from housekeeping or constitutively expressed genes.

6. A method of expression according to any one of claims 2 to 4, wherein the regulatory sequences are derived from tissue-specific or inducible genes.

7. A method of expression according to any one of claims 2 to 4, wherein the regulatory sequences are matrix attachment regions (MAR) or originate from these.

8. A method of expression according to any one of claims 2 to 4, wherein the regulatory sequences are locus control regions (LCR) or originate from these.

9. A method of expression according to claim 1, wherein the exogenous sequences to be expressed in combination with sequences

Mammalian cells are transmitted, which enable a homologous recombination with endogenous 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.

10. A method of expression according to claim 9, wherein the sequences transmitted in combination with the exogenous sequences to be expressed comprise R band sequences.

11. A method of expression according to claim 9 or 10, wherein the in

Combination with the exogenous sequences to be expressed include aluminum sequences.

12. A method for the expression of exogenous sequences in mammalian cells, characterized in that the sequences are specifically integrated into DNA of the inactive compartment of the nucleus of the mammalian cells.

13. A method for expression according to claim 12, wherein the exogenous sequences to be expressed in combination with regulatory sequences are transferred to mammalian cells which are stable expression after Ensure 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.

14. A method for expression according to claim 13, wherein the regulatory sequences comprise promoter and / or enhancer sequences or other sequences with a regulatory function.

15. A method of expression according to claim 13 or 14, wherein the regulatory sequences are of viral origin.

16. A method of expression according to any one of claims 13 to 15, wherein the regulatory sequences are derived from tissue-specific or inducible genes.

17. A method for expression according to any one of claims 13 to 15, wherein the regulatory sequences are derived from housekeeping or constitutively expressed genes.

18. A method of expression according to any one of claims 13 to 15, wherein the regulatory sequences are matrix attachment regions (MAR) or originate from these.

19. A method of expression according to any one of claims 13 to 15, wherein the regulatory sequences are locus control regions (LCR) or originate from these.

20. A method of expression according to claim 12, wherein the exogenous sequences to be expressed in combination with sequences are transferred to mammalian cells which have a homologous recombination with enable endogenous sequences in the inactive compartment of the nucleus of the mammalian cell and thus reduce the risk of insertional mutagenesis.

21. A method of expression according to claim 20, wherein the transferred in combination with the exogenous sequences to be expressed

Sequences include G-band sequences and / or C-band sequences.

22. A method for expression according to claim 20 or 21, wherein the sequences transmitted in combination with the exogenous sequences to be expressed comprise LINE sequences and / or satellite DNA sequences.

23. A method for expression according to any one of the preceding claims, wherein the exogenous sequences are genes from viruses, archaebacteria, bacteria, fungi, plants, invertebrates, vertebrates, especially mammals.

24. A method for identifying regulatory sequences or sequences suitable for homologous recombination with endogenous sequences, e.g. Alu 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 fusion of regulatory sequences or sequences suitable for homologous recombination with endogenous sequences, e.g. Alu sequences, with exogenous sequences to be expressed on mammalian cells and b) localization of the integrated fusion in the cell nucleus and / or on the

Chromosomes using cell biological and / or cytogenetic methods.

25. A method for identifying sequences suitable for homologous recombination with endogenous sequences, for example LINE sequences, which contain a Promote stable integration of sequences to be expressed in sequences of the inactive compartment of the nucleus of a mammalian cell, comprising the steps of: a) transfer of a fusion of regulatory sequences which allow expression even after recombination with sequences of the inactive compartment and for homologous recombination with endogenous sequences suitable sequences, for example LINE sequences, with exogenous sequences to be expressed on mammalian cells and b) localization of the integrated fusion in the cell nucleus and / or on the chromosomes by means of cell biological and / or cytogenetic methods.

26. Method for localizing endogenous sequences in the active compartment or in the inactive compartment of the nucleus of a mammalian cell by means of cell biological methods.

27. 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.

28. Use of the method according to one of claims 1 to 23 in the production of transgenic animals.

29. Use according to claim 28, wherein the transgenic animal has expression of a transgenic product in the milk of the animal.

30. Use according to claim 28 or 29 for the production of a biomedical product.

31. Use according to claim 28 for the production of cells, tissues and organs for the purposes of transplantation.

32. Use according to claim 28 for the targeted change of properties, e.g. of hair, milk composition, height or weight.

33. Use of the method according to one of claims 1 to 23 in gene therapy.

34. Use of the method according to claim 24 for the production of vectors, in particular vectors for gene therapy, for a

Process according to claim 1 are suitable.

35. Use of the method according to claim 24 for screening and for the development of corresponding vectors in connection with the production of transgenic animals.

36. Use of the method according to claim 25 for the production of vectors, in particular vectors for gene therapy, which are suitable for a method according to claim 12.