ES2400296T3 - Transfer and expression of highly efficient genes in mammalian cells by a multiple transfection method of sequences of regions of fixation to the matrix - Google Patents

Abstract

An isolated and purified DNA sequence that has an activity that increases protein production, characterized in that said DNA sequence comprises a) at least one curved DNA element, where the curved DNA element contains at least 33% of TA dinucleotide and / or at least 33% of the AT dinucleotide in a contiguous 100 base pair span, b) at least one binding site for a DNA binding protein, and because it is a MAR nucleotide sequence having the sequence SEQ ID No 27, or one of its complementary sequences, or a sequence that has an identity of at least 90% with said sequence SEQ ID No 27.

Description

Transfer and expression of highly efficient genes in mammalian cells by a method of multiple transfection of sequences from regions of fixation to the matrix.

FIELD OF THE INVENTION

The present invention relates to isolated and purified DNA sequences having activity that increases protein production and more specifically to the use of matrix-binding regions (hereinafter abbreviated MAR by the English expression Matrix Attachment Regions) to increase protein production activity in a eukaryotic cell. Also described is a method for identifying such active regions, in particular MAR nucleotide sequences and the use of these characterized active MAR sequences in a novel multiple transfection method.

BACKGROUND OF THE INVENTION

Currently, the model of organization of looped domains of eukaryotic chromosomes is widely accepted (Boulikas T, "Nature of DNA sequences at the attachment regions of genes to the nuclear matrix", J. Cell Biochem.,

52: 14-22, 1993). According to this model, chromatin is organized in loops that span 50-100 kb fixed to the nuclear matrix, a protein lattice made up of ribonucleoproteins (RNP) and other non-histonic proteins (Bode J, Stengert-Iber M, Kay V, Schalke T and Dietz-Pfeilstetter A, Crit. Rev. Euk. Gene Exp., 6: 115-138, 1996).

The DNA regions attached to the nuclear matrix are called SAR (for the Scaffold Attachment Region) or MAR, for regions of fixation to the framework (during the metaphase) or to the matrix (interface), respectively (Hart C and Laemmli U. (1998), "Facilitation of chromatin dynamics by SARs", Curr. Opin. Genet. Dev. 8, 519-525.)

In this sense, these regions can define the boundaries of the independent chromatin domains, in such a way that only the enveloping cis-regulatory elements control the expression of the genes in the domain.

However, its ability to fully protect a chromosomal locus from nearby chromatin elements and thereby confer position-independent gene expression on them, has not been observed in stably transfected cells (Poljak L, Seum C, Mattioni T and Laemmli U. (1994) "SARs stimulate but do not confer position independent gene expression", Nucleic Acids Res 22, 4386-4394). On the other hand, MAR (or S / MAR) sequences have been shown to interact with enhancers to increase accessibility to local chromatin (Jenuwein T, Forrester W, Fernandez-Herrero L, Laible G, Dull M, and Grosschedl R . (1997) "Extension of chromatin accessibility by nuclear matrix attachment regions", Nature, 385, 269-272). Specifically, MAR elements can enhance heterologous gene expression in cell culture lines (Kalos M and Fournier R (1995) "Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain", Mol. Cell Biol. , 15,198-207), transgenic mice (Castilla J, Pintado B, Sola I, Sánchez-Morgado J, and Enjuanes L (1998) "Engineering passive immunity in transgenic mice secreting virus neutralizing antibodies in milk", Nat. Biotechnol., 16, 349-354) and vegetables (Allen G, Hall GJ, Michalowski S, Newman W, Spiker S, Weissinger A, and Thompson W (1996), "High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco ", Plant Cell 8, 899-913). The usefulness of MAR sequences for developing improved vectors for gene therapy is also recognized (Agarwal M, Austin T, Morel F, Chen J, Bohnlein E, and Plavec I (1998), "Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells ", J. Virol 72, 3720-3728).

Recently, chromatin structure modifying sequences including MARs have been shown, as illustrated by MAR at the 5 'end of chicken lysozyme, to be able to significantly enhance expression of reporter genes in mixtures of Chinese Hamster Ovary Cells (abbreviated CHO for Chinese Hamster Ovary) (Zahn-Zabal M, et al., "Development of stable cell lines for production or regulated expression using matrix attachment regions", J. Biotechnol, 2001 , 87 (1):

p. 29-42). This property was used to increase the proportion of high production clones, thus reducing the number of clones that need to be screened. These benefits have been observed both in constructs with MARs flanking the transgenic expression cassette, and when constructs are co-transfected with MARs into a separate plasmid. However, the expression levels in co-transfection together with the MARs were not as high as those observed for a construct in which two MARs delimit the transgene expression unit. A third preferred process was shown which was transfection of transgenes with MARs both bound to the transgene and into a separate plasmid (Girod et al., Pending publication). However, a persistent limitation of this technique is the amount of DNA that can be transfected per cell. Many multiple transfection protocols have been developed in order to achieve high transfection efficiency to characterize the function of the genes of interest. The protocol applied by Yamamoto et al., 1999 ("High efficiency gene transfer by multiple transfection protocol", Histochem. J. 31 (4), 241-243) leads to a transfection efficiency of approximately 80% after 5 events of transfection, whereas the conventional transfection protocol only achieved a rate <40%. Although this technique may be useful if you want to increase the proportion of cells that are expressed, it does not lead to cells with higher intrinsic productivity. Consequently, it cannot be used to generate highly productive monoclonal cell lines. Therefore, the technique described above has two main drawbacks:

i) this technique does not generate a homogeneous population of transfected cells, since it cannot favor the integration of more gene copies or direct the transgenes to favorable chromosomal loci,

ii) the use of the same selectable marker in multiple transfection events does not allow the selection of double or triple transfected cells.

In patent application WO02 / 074969, the utility of MARs for the development of stable eukaryotic cell lines has also been demonstrated. However, this application describes neither the conserved homology of the MAR DNA element nor any technique to predict the ability of a DNA sequence to be a MAR sequence.

Indeed, no net MAR consensus sequence has been found (Boulikas T, "Nature of DNA sequences at the attachment regions of genes to the nuclear matrix", J. Cell Biochem., 52: 14-22, 1993) but that by evolution, it seems that the structure of these sequences is functionally conserved in eukaryotic genomes, since the MAR of animals can bind to the nuclear frameworks of plants and vice versa (Mielke C, Kohwi Y, Kohwi-Shigematsu T and Bode J, "Hierarchical binding of DNA fragments derived from scaffold-attached regions: correlation of properties in vitro and function in vivo", Biochemistry, 29: 7475-7485, 1990).

The identification of MARs by biochemical studies is a long and unpredictable process; Different results may be obtained depending on the assay (Razin SV, "Functional architecture of chromosomal DNA domains", Crit. Rev. Eukaryot. Gene Expr., 6: 247-269, 1996). Considering the enormous number of MARs expected in a eukaryotic genome and the number of sequences obtained from genome projects, a tool capable of filtering potential MARs in order to carry out specific experiments would be very useful.

Currently, two different predictive tools for MAR are available online. The first is the MAR-Finder software (http: // futuresoflorg / MarFinder; Singh GB, Kramer JA and Krawetz SA, "Mathematical model to predict regions of chromatin attachment to the nuclear matrix". Nucleic Acid Research, 25: 1419- 1425, 1997) is based on an identified set of patterns in the various MARs and a statistical analysis of the coexistence of these patterns. The predictions by the MAR-Finder program depend on the context of the sequence, which means that the predicted MARs depend on the context of the analyzed sequence. The other prediction software, the SMARTest program, (hftp: //www.genomatix.de; Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, "In silico prediction of scaffold / matrix attachment regions in large genomic sequences ", Genome Research, 12: 349-354, 2001), uses weighted matrices derived from experimentally identified MARs. The SMARTest program is said to be suitable for large-scale analysis. But really, in addition to its relatively low specificity, the amount of hypothetical MARs is greatly increased when a large-scale analysis is performed with it and there being no way to increase its specificity to restrict the number of hypothetical MARs, the SMARTest program is almost useless for Screen powerful MARs from large DNA sequences.

There are also some other computer programs, not available on the Internet; they are also based on the frequency of MAR residues (MRS criterion; Van Drunen CM et al., "A bipartite sequence element associated with matrix / scaffold attachment regions", Nucleic Acids Res, 27: 2924-2930, 1999), (ChrClass ; Glazko GV et al., "Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix", Biochim. Biophys. Acta, 1517: 351-356, 2001) or are based on the identification of duplex sites of Stress-induced DNA (abbreviated SIDD by the English expression Stress-Induced DNA Duplex); Benham C and al., "Stressinduced duplex DNA destabilization in scaffold / matrix attachment regions", J. Mol. Biol., 274: 181-196, 1997). However, their suitability for analyzing entire genome sequences is unknown, and it has not been reported whether these tools can allow the identification of sequences that increase protein production.

Furthermore, due to the relatively low specificity of these computer programs (Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, "In silico prediction of scaffold / matrix attachment regions in large genomic sequences", Genome Research, 12: 349-354, 2001), the amount of hypothetical MAR identified in genomes makes them rapidly unmanageable when performing large-scale analyzes, especially if most of them have little or no activity in practice. Therefore, having no way of increasing the prediction specificity to restrict the number of hypothetical MARs, many of the available programs are practically useless to identify powerful genetic elements in view of the effective increase in the production of recombinant proteins.

Since all the previous available prediction methods have some drawbacks that prevent large-scale analysis of genomes to identify with certainty new and powerful MAR, the purpose of this description is: 1) to understand the functional characteristics of MAR that allow to improve the recombinant protein expression; 2) get a new bioinformatics tool that compiles the structural characteristics of MARs as a prediction of their function, in order to 3) carry out large-scale analyzes of genomes to identify new and more powerful MARs and, finally 4) demonstrate better efficacy for increase the production of recombinant proteins from eukaryotic cells or organisms when recently identified MAR sequences are used.

SUMMARY OF THE INVENTION

An object of the invention is to increase the production of recombinant proteins from eukaryotic cells or organisms using a DNA sequence that has an activity that increases protein production.

Another object of the invention is to provide a multiple transfection method to increase the production of recombinant proteins in eukaryotic cells.

These objects are achieved by an isolated and purified DNA sequence, by the use of said isolated and purified DNA sequence, by a method to transfect a eukaryotic host cell, by a process for the production of a protein, by a eukaryotic host cell. transfected, by a mixture or kit for cellular transfection and by a non-human transgenic organism, according to the independent claims.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows distribution graphs of the MAR and non-MAR sequences. Histograms are graphs of density (relative frequency divided by the width of the rectangle) relative to the score of the observed parameter. The density histogram of human MARs in the SMARt DB database is shown in black, while the density histogram of human chromosome 22 is gray.

Fig. 2 shows scatter diagrams of the four different criteria used by the SMAR Scan® program and the AT content with human MARs of the SMARt DB.

Fig. 3 shows the distribution graphs of the MAR sequences by organism. The sequences of the MARs of the SMARt DB from other organisms were recovered and analyzed. The density distributions of the MAR sequences in mice, chicken, sorghum bicolor and humans are jointly represented.

Fig. 4 shows the SMAR Scan® predictions of human chromosome 22 and shuffled chromosome 22. Upper graph: average number of coincidences obtained by the SMAR Scan® program with five chromosomes: “rubbled”, “scrambled”, shuffled in non-overlapping windows of 10 bp, model of Markov chains of order 1 and with natural chromosome 22. Lower graph: average number of MAR predicted by the SMAR Scan® program on five chromosomes: “rubbled”, “scrambled”, shuffled in non-overlapping windows of 10 bp, model of Markov chains of order 1 and with natural chromosome 22.

Fig. 5 shows the dissection of the ability of the chicken 5'-MAR lysozyme gene to stimulate transgene expression in CHO-DG44 cells. Fragments B, K and F show the highest capacity to stimulate transgene expression. The indicated relative resistance of the elements was based on the number of cells with high expression activity.

Fig. 6 shows the effect of serial deletions at the 5 'end (top) and at the 3' end (bottom) of 5'-MAR on loss of ability to stimulate transgene expression. The transition from increased activity to decreased activity coincides with fragments B, K and F.

Fig. 7 shows which portions of fragment F significantly stimulate transgene expression. The F fragment regions indicated by the light gray arrow were multimerized, inserted into pGEGFP Control and transfected into CHO cells. The element that exhibits the most activity is located in the central part of the element and corresponds to the fragment FIll (minimum MAR indicated by the black bar). Furthermore, an enhancing activity is located in the 3 ′ flanking part of the FIll fragment (MAR enhancer indicated by the dark gray bar).

Fig. 8 shows a location map of various DNA sequence residues in cLysMAR. Fig. 8 (B) represents a location map of various DNA sequence residues in cLysMAR. The vertical lines represent the position of the computer predicted sites or sequence residues along the 3034 base pairs of cLysMAR and their active regions, represented in Fig. 5. The putative transcription factor sites (MEF2 05, Oct-1, USF-02, GATA, NFAT) for activators and (CDP, SATB1, CTCF, ARBP / MeCP2) for transcriptional repressors, were identified using the Matinspector program (Genomatix), and the CpG islands were identified with the CPGPLOT program. Residues to which MAR elements have previously been associated are indicated in black and include CpG dinucleotide and CpG island, unwinding residues (AATATATT and AATATT), poly As and Ts, poly Gs and Cs, binding sites for Drosophila topoisomerase II (GTNWAYATTNATTNATNNR) that had the same identity as the 6 bp nucleus and binding sites for high mobility group I (HMG-I / Y) proteins. Other structural residues include sites for nucleosome binding and unfavorable for nucleosome binding and a residue intended to release the superhelical chain of DNA. Fig. 8 (A) represents the comparison of the ability of portions of cLysMAR to activate transcription with prediction scoring profiles with the MAR-Finder program. The top diagram shows the activity of MAR fragments as in Fig. 5, while the center and bottom curves show the potential predicted by the MAR-Finder program for MAR activity and for curved DNA structures, respectively.

Fig. 9 shows the correlation between the physicochemical properties of DNA and the activity of MAR. Fig. 9 (A) depicts the profiles of the melting temperature, the double helix curvature, the depth of the major groove and the width of the minor groove of the 5'-MAR DNA that were determined using the algorithms de Levitsky et al., (Levitsky VG, Ponomarenko MP, Ponomarenko JV, Frolov AS, Kolchanov NA, "Nucleosomal DNA property database", Bioinformatics, 15; 582-592, 1999). The most active fragments B, K and F represented at the top are those shown in Figure 1. Fig. 9 (B) represents the magnification of the data presented in panel A that represents the map of fragment F aligned with the trace corresponding to the melting temperature (upper curve) and DNA curvature (lower curve). The position of the most active FIB fragment and the protein binding site for specific transcription factors are indicated.

Fig. 10 shows the distribution of the sites of binding to the putative transcription factor in 5'-cLysMAR. The large arrows indicate the position of the core unwinding elements (abbreviated CUE, for the English expression Core Unwinding Elements) identified with the SMAR Scan® program.

Fig. 11 shows the assembly scheme of various portions of the MAR. The indicated portions of cLysMAR were amplified by PCR, inserting Bglll-BamHl linker elements at each end, and assembled to generate the depicted composite elements. For example, the top construct consists of the assembly of all flanking and CUE sequences in their original position except that the BgII-BamHII connecting sequences separate each element.

Fig. 12 depicts the plasmid maps.

Fig. 13 shows the effect of re-transfecting primary transfectants on the expression of the green fluorescent protein (abbreviated hereafter GFP, for the English expression Green Fluorescent Protein). Cells (CHO-DG44) were co-transfected with pSV40EGFP (left tube) or pMAR-SV40EGFP (center tube) and pSVneo as resistance plasmid. Cells transfected with pMAR-SV40EGFP were transfected 24 hours later with the same plasmid and a different selected plasmid, pSVpuro (right tube). After a two-week selection, the phenotype of the stably transfected cell population was analyzed by FACS.

Fig. 14 shows the effect of multiple loading of plasmid containing MAR. Secondary transfectants pMAR-SV40EGFP / pMAR-SV40EGFP were used in a third cycle of transfection at the end of the selection process. Tertiary transfection was performed with pMAR or pMAR-SV40EGFP to obtain tertiary transfectants. After 24 hours, cells were re-transfected with each plasmid, resulting in quaternary transfectants (see Table 4).

Fig. 15 shows the comparative action of the SMAR prediction algorithms illustrated by the WP18A10A7 region. (A) Analysis by the SMAR Scan @ program was performed with the default settings. (B) The analysis of the SIDD (upper curve and lateral scale on the left hand) and the fixation of various DNA fragments to the nuclear matrix in vitro (bar graph, lateral scale on the right hand) were taken from Goetze et al., (Goetze S, Gluch A, Benham C, Bode J, "Computational and in vitro analysis of destabilized DNA regions In the interferon gene cluster: potential of predicting functional gene domains." Biochemistry, 42: 154-166, 2003).

Fig. 16 represents the results of a protocol similar to gene therapy using MARs. The group of mice injected with MAR-Network, induced since the beginning of the experiment, presents a better induction of the hematocrit compared to the mice injected with the original Network system without MAR. After 2 months, hematocrit in the "MAR containing group" has higher values (65%) than normal hematocrit levels (45-55%).

Fig. 17 represents the scatterplot for the 1757 S / MAR sequences of the percentages of AT (upper) and TA (lower) dinucleotides versus the predicted DNA curvature calculated by the SMAR Scan® program.

Fig. 18 represents the distribution graphs of the percentages of dinucleotides in the 1757 non-S / MAR sequences.

Fig. 19 presents the effect of various elements of S / MAR on the production of the recombinant green fluorescent protein (GFP). Populations of CHO cells transfected with a GFP expression vector containing a MAR element, as indicated, were analyzed by a fluorescence activated cell sorter (FACS®) and typical profiles are shown. The profiles present the number of cells as a function of the GFP fluorescence levels.

Fig. 20 represents the effect of hematocrit induction in mice injected with MAR-Network.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated and purified DNA sequence having an activity that increases protein production, characterized in that said DNA sequence comprises: a) at least one curved DNA element and b) at least one binding site for a protein that binds to DNA.

Certain DNA sequences are known to form a "relatively static curve", in which DNA follows a particular three-dimensional path. Therefore, instead of having the normal ("linear") B-DNA conformation, the DNA segment can form a flat curve also defined as curved DNA (Marini, et al., 1982 "Bent helical structure in kinetoplast DNA", Proc. Natl. Acad. Sci. USA, 79: 7664-7664).

Surprisingly, the inventors have shown that the curved DNA element of an isolated and purified DNA sequence having an activity that increases protein production generally contains at least 10% of the TA dinucleotide and / or at least 12% of the AT dinucleotide in a span of 100 contiguous base pairs. The curved DNA element of a DNA sequence according to the invention contains at least 33% of the TA dinucleotide and / or at least 33% of the AT dinucleotide over a span of 100 contiguous base pairs. These data have been obtained by the method described below.

The isolated and purified DNA sequence according to the invention comprises a MAR nucleotide sequence containing the sequence SEQ ID No. 27.

Sequences complementary to the aforementioned SEQ ID No. 27 sequence, which can be produced using PCR or any other means, are also encompassed by the present invention.

Also included in the present invention are sequences that share at least a nucleotide length of 90% of the respective SEQ ID No. 27 sequence. These sequences can be used as long as they have the same properties as the natural sequence from which they come.

The present description also includes variants of the aforementioned SEQ ID No. 1 to 27 sequences and the cLysMAR element or fragment, i.e. nucleotide sequences that vary relative to the reference sequence by conservative nucleotide substitutions, wherein one or more nucleotides are replaced by others with the same characteristics.

The sequences SEQ ID No 1 to 23 shown in this description have been identified by scanning human chromosomes 1 and 2 using the SMAR Scan @ program, which shows that it is possible to identify new MAR sequences using the tools described below, while sequences SEQ ID No 24 to 27 have been identified by scanning the entire human genome, using the combined SMAR Scan @ method.

In a first stage, complete chromosomes 1 and 2 were screened to identify the curved DNA element as the region corresponding to curvature, depth of major groove, width of maximum minor groove, and minimum melting temperature, as shown in Figure 3. In a second step, this collection of sequences was scanned to determine the binding sites of regulatory proteins, such as SATB1, GATA, etc., as shown in Figure 8B, obtaining the sequences SEQ ID 1 -2. 3. Furthermore, sequences 21-23 were shown to be located close to a known gene from the human genome database.

The sequences SEQ ID No 24 to 27 were obtained by scanning the human genome according to the combined method and were selected as examples among the 1757 elements of MAR thus detected.

The molecular chimeras of the MAR sequences are also part of the present description. By molecular chimera is meant a nucleotide sequence that can include a functional portion of an MAR element and that will be obtained by molecular biology methods known to those skilled in the art.

Particular combinations of elements or fragments of MAR or its sub-portions are also part of the present description. These fragments can be prepared by a variety of methods known in the art. These methods include, but are not limited to, restriction enzyme digestion and fragment recovery, chemical synthesis, or polymerase chain reactions (PCR).

Accordingly, particular combinations of elements or fragments of sequences SEQ ID No. 1 to 27 and elements or fragments of cLysMAR are also part of the present description, depending on the functional results that are obtained. Elements of cLysMAR are for example the B, K and F regions described in WO 02/074969. Preferred cLysMAR elements used in the present disclosure are regions B, K and F. Only one element or multiple copies thereof could be used or different elements (multimerized elements) could be used (see Fig. 8 A).

Fragment is understood to mean a portion of the respective nucleotide sequence. Fragments of a MAR nucleotide sequence can retain biological activity and therefore bind to purified nuclear matrices and / or alter the expression patterns of coding sequences operably linked to a promoter. Fragments of a MAR nucleotide sequence can vary between nucleotides of at least about 100 to 1000 bp, preferably from about 200 to 700 bp, more preferably from about 300 to 500 bp. Also part of the present disclosure includes combinations of fragments, having the same number of nucleotides present in a synthetic MAR sequence consisting of natural MAR elements and / or fragments. The fragments are preferably assembled by connecting sequences. The preferred linker is BgIll-BamHl.

An "element" is a conserved nucleotide sequence that has common functional properties (ie, binding sites for transcription factors) or structural features (ie, curved DNA sequence).

"Protein production enhancing activity" refers to an activity of the isolated, purified DNA sequence defined as follows: after it has been introduced under suitable conditions into a eukaryotic host cell, the sequence is capable of increasing levels of protein production in a cell culture compared to a culture of transfected cells without such a DNA sequence. Generally, the increase is 1.5 to 10 times, preferably 4 to 10 times. This corresponds to a specific cell production or productivity rate of at least 10 pg per cell per day (see Example 11 and Fig. 13).

As used herein, the following definitions are provided in order to facilitate understanding of this invention.

"Chromatin" is the protein and nucleic acid material that makes up the chromosomes of a eukaryotic cell, and refers to DNA, RNA, and associated proteins.

A "chromatin element" means a nucleic acid sequence on a chromosome that has the property of modifying the structure of chromatin when it is integrated into said chromosome.

"Cis" refers to the placement of two or more elements (such as chromatin elements) on the same nucleic acid molecule (such as the same vector, plasmid, or chromosome).

"Trans" refers to the placement of two or more elements (such as chromatin elements) on two or more different nucleic acid molecules (such as on two vectors or two chromosomes).

Chromatin modifying elements that are potentially capable of overcoming position effects and are therefore of interest for the development of stable cell lines include border elements (abbreviated BE, for Boundary Elements), regions of fixation to the matrix (MAR), locus control regions (abbreviated LCR, for the English expression Locus Control Regions) and ubiquitous elements that open chromatin (abbreviated UCOE, for the English expression Universal / Ubiquitously Chromatin Opening Elements).

Edge elements ("BE") or insulating elements, in many cases define limits on chromatin (Bell A and Felsenfeld G. 1999; "Stopped at the border: boundaries and insulators", Curr. Opin. Genet. Dev. , 9, 191-198) and may play a role in defining a transcriptional domain in vivo. BEs lack intrinsic promoter / enhancer activity, but are instead believed to protect genes from the transcriptional influence of regulatory elements on the surrounding chromatin. Generally, enhancer blocking analysis is used to identify the insulating elements. In this assay, the chromatin element is placed between an enhancer and a promoter and enhancer-activated transcription is measured. Isolator elements have been shown to be able to protect stably transfected reporter genes against positional effects in Drosophila, yeast, and mammalian cells. They have also shown to increase the proportion of transgenic mice with inducible expression of transgenes.

Locus control regions ("CSF") are cis-regulatory elements required for initial activation of locus chromatin and subsequent gene transcription in their natural positions (Grosveld, F. 1999, "Activation by locus control regions?" Curr . Opin. Genet. Dev., 9, 152-157). The activating function of CSFs also allows the expression of a coupled transgene in the appropriate tissue in transgenic mice, regardless of the site of integration in the host genome. Although CSFs generally confer tissue-specific levels of expression in the genes to which they bind, effective expression has been reported in almost all tissues in mice transgenic for a truncated receptor CSF from human T lymphocytes and a CSF from the protein rat liver activator (abbreviated LAP for the English expression Liver Activating Protein). The most widely characterized CSF is that of the globin locus. Its use in vectors for gene therapy of sickle cell diseases and �-thalassemias is currently being evaluated.

"MARs", according to a widely accepted model, can mediate the anchoring of a specific DNA sequence in the nuclear matrix, generating chromatin loop domains that extend out of the heterochromatin nuclei. Although the MARs do not contain any obvious consensus or recognizable sequence, it seems that their most consistent characteristics are a high global content of A / T and predominant C bases in a chain (Bode J, Schlake T, Ríos Ramírez M, Mielke C, Stengart M, Kay V and KlehrWirth D, "Scaffold / matrixattached regions: structural properties creating transcriptionally active loci", Structural and Functional Organization of the Nuclear Matrix: International Review of Citology, 162A: 389-453, 1995). These regions have a propensity to form curved secondary structures that may be predisposed to chain separation. They are often referred to as base mismatch regions (BUR for short), and contain a core unwinding element (CUE) that could represent the nucleation point of chain separation (Benham C and al., "Stress induced duplex DNA destabilization in scaffold / matrix attachment regions", J. Mol. Biol., 274: 181-196, 1997). Various residues of simple AT-rich sequences have frequently been found in MAR sequences, but in most cases their functional importance and potential mode of action are unclear. These residues include box A (AATAAAYAAA), box T (TTWTWTTWTT), DNA unwinding residues (AATATATT, AATATT), SATB1 binding sites (box H, A / T / C26) and consensus sites of topoisomerase II for vertebrates (RNYNNCNNG-YNGKTNYNY) or Drosophila (GTNWAYATTNATNNR).

Universal chromatin opening elements ("UCOE", also known as "ubiquitous chromatin opening elements") have been described in WO 00/05393.

An "enhancer" is a nucleotide sequence that acts to enhance gene transcription regardless of the identity of the gene, the position of the sequence relative to the gene, or the orientation of the sequence. The vectors of the present invention optionally include enhancers.

A "gene" is a sequence of deoxyribonucleotides (DNA) that encodes a given mature protein. As used herein, the term "gene" should not include untranslated flanking regions, such as RNA transcription initiation signals, polyadenylation addition sites, promoters, or enhancers.

A "product gene" is a gene that encodes a protein product that has desirable characteristics, such as utility in diagnosis or therapy. A product gene includes, for example, structural genes and regulatory genes.

A "structural gene" refers to a gene that encodes a structural protein. Examples of structural genes include, but are not limited to, cytoskeletal proteins, extracellular matrix proteins, enzymes, nuclear pore proteins, and nuclear framework proteins, channels and transporters, contractile proteins, and chaperones. Preferred structural genes encode antibodies or antibody fragments.

A "regulatory gene" refers to a gene that encodes a regulatory protein. Examples of regulatory proteins include, but are not limited to, transcription factors, hormones, growth factors, cytokines, signal transduction molecules, oncogenes, proto-oncogenes, transmembrane receptors, and protein kinases.

"Orientation" refers to the order of nucleotides in a given DNA sequence. For example, an inverted orientation of a DNA sequence is one in which the order of the sequence is reversed 5 'to 3' relative to another sequence compared to a reference point in the DNA from which the sequence. Such reference points may include the direction of transcription of other specified DNA sequences in the source DNA and / or the origin of replication of replicable vectors containing the sequence.

"Eukaryotic cell" refers to any mammalian or non-mammalian cell derived from a eukaryotic organism. As a non-limiting example, any eukaryotic cell that is capable of maintaining itself under the conditions of cell culture and subsequently transfected would be included in this invention. Especially preferred cell types include, for example, stem cells, embryonic stem cells, Chinese hamster ovary (CHO) cells, COS, BHK21, NIH3T3, HeLa, C2C12, cancer cells, and differentiated primary cells.

or undifferentiated. Other suitable host cells are known to those skilled in the art.

The terms "host cell" and "recombinant host cell" are used interchangeably herein to indicate a eukaryotic cell into which one or more vectors of the invention have been introduced. It is to be understood that said expressions refer not only to the particular object cell, but also to the progeny or potential progeny of said cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may, in effect, not be identical to the parent cell, but is also included within the scope of the expressions used herein.

The terms "introducing purified DNA into a eukaryotic host cell" or "transfection" mean any process in which extracellular DNA, with or without accompanying material, enters a host cell. The term "transfected cell" means the cell into which the extracellular DNA has been introduced and therefore houses the extracellular DNA. DNA could be introduced into the cell so that the nucleic acid is replicable as a chromosomal component or as an extrachromosomal element.

"Promoter" as used herein refers to a nucleic acid sequence that regulates the expression of a gene.

"Co-transfection" means the process of transfecting a eukaryotic cell with more than one exogenous gene or vector or plasmid, foreign to the cell, one of which may confer on the cell a selectable phenotype.

The isolated and purified DNA sequence having activity that increases protein production also comprises, in addition to one or more elements of curved DNA, at least one binding site for a protein that binds to DNA.

Generally the protein that binds to DNA is a transcription factor. Examples of transcription factors are the group consisting of proteins with the polyQpolyP domain. Another example of a transcription factor is a transcription factor selected from the group comprising SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C / EBP, CREBP1, FOX, Freac7, HFH1, HNF3alfa, Nkx25, POU3F2, Pit1, TTF1, XFD1, AR, C / EBPgamma, Cdc5, FOXD3, HFH3, HNF3beta, MRF2, Oct1, POU6F1, SRF, V $ MTD2, B CR3, Cdx2, FOXJ2, HFL, HP1, Myc, PBX, Pax3, TEF, VBP, XFD3, Bm2, COMP1, Evil, FOXP3, GATA4, HFN1, Lhx3, NKX3A, POU1F1, Pax6, TFIIA or a combination of two or more of these transcription factors. Most preferred are proteins with the SATB1, NMP4, MEF2 and polyQpolyP domains.

For example, SATB1, NMP4 and MEF2 are known to regulate the development and / or expression of tissue-specific genes in mammals. These transcription factors have the ability to alter the geometry of DNA and, conversely, binding to DNA as an allosteric ligand modifies its structure. Recently, SATB1 was found to form a cage-like structure that circumscribes heterochromatin (Cai S, Han HJ, and Kohwi-Shigematsu T, "Tissue-specific nuclear architecture and gene expression regulated by SATB1" Nat. Genet., 2003. 34 (1): p. 42-51).

Another part of the present description is an isolated and purified cLysMAR element and / or fragment, one of its complementary sequences, one of its parts that shares a nucleotide length of at least 70%, one of its molecular chimeras and one of their combinations and variants.

The cLysMAR element and / or fragment may consist of at least one nucleotide sequence selected from regions B, K, and F.

An additional part of the present description is a synthetic MAR sequence comprising elements and / or fragments of natural MAR assembled between connecting sequences.

The synthetic MAR sequence can comprise an element and / or fragment of cLysMAR from one of its complementary sequences, one of its parts that shares at least a nucleotide length of 70%, one of its molecular chimeras and one of its combinations and variants . Also preferably, the linker sequences are the Bglll-BamHl linker.

Another aspect of the disclosure is to provide a method for identifying a MAR sequence using a bioinformatics tool that comprises calculating values of one or more characteristics of DNA sequences corresponding to DNA curvature, major groove depth, and Potential minor groove width and melting temperature. Preferably, the identification of one or more characteristics of the DNA sequences further comprises one more characteristic of the DNA sequences corresponding to the binding sites for the DNA-binding proteins, which is also calculated by this method.

Preferably, the profiles or the weighted matrices of said bioinformatics tool are based on the recognition of dinucleotides.

The bioinformatics tool used for the present method is preferably the SMAR Scan @ program, which contains algorithms developed by Gene Express ((http://srs6.bionet.nsc.ru/srs6bin/cgi-bin/wgetz?-e++ FEATURES -SiteID: 'nR']) and is based on the work of Levitsky et al., 1999. These algorithms recognize profiles, based on weighted dinucleotide matrices, to calculate the theoretical values of the conformational and physicochemical properties of DNA.

Preferably, the SMAR Scan @ program uses the four theoretical criteria also designated as characteristics of the DNA sequences corresponding to the curvature of the DNA, the depth of the largest groove and the width of the smallest potential groove and the melting temperature in all possible combinations, using variable size scan windows (see Fig. 3). For each function used, an exclusion value must be set. The program matches each time the calculated score for a given region is above the adjusted exclusion value for all chosen criteria. Two data output modes are available to handle matches, the first (called "profile-like") simply presents all the positions of the matches in the query sequence and their corresponding values for the different criteria chosen. The second mode (called "contiguous matches") only produces the positions of various contiguous matches and their corresponding sequences. For this mode, the minimum number of contiguous matches is another exclusion value that can be set, again with an adjustable window size. This second mode is the default mode of the SMAR Scan® program. Actually, from a semantic point of view, a coincidence is considered a nucleus unwinding element (CUE) and a grouping of CUE accompanied by groupings of binding sites for the corresponding proteins is considered a MAR. Therefore, the SMAR Scan @ program only considers multiple contiguous matches as a potential MAR.

To fine-tune the default exclusion values for the four theoretical structural criteria, experimentally validated MARs from the SMARt DB (http://transfac.gbf.de/-SMARt DB) were used. The inventors retrieved and analyzed all human MAR sequences from the database with the SMAR Scan® program using the "profile-like" mode with all four criteria and without adjusting the exclusion value. This allowed the adjustment of each function for each position of the sequences. Based on these data, the distribution of each criterion was then calculated (see Figs. 1 and 3).

The default SMAR Scan® program exclusion values for curvature, depth of major groove, and width of minor groove were adjusted to the 75th quantile mean and median. For the melting temperature, the default exclusion value should be set to the 75th quantile. The minimum length for the "contiguous matches" mode should be set to 300 because it is assumed to be the minimum length of a MAR (see Fig. 8 and 9). However, one skilled in the art could determine the exclusion values of the above criteria for a given organism with minimal experimentation.

Preferably, the DNA curvature values are between 3 and 5 ° (radial degree). More preferably they are located between 3.8 and 4.4 °, corresponding to the minor peak in Fig. 1.

Preferably, the values of the depth of the largest groove are between 8.9 and 9.3 Å (Angstrom) and the values of the width of the smallest groove are between 5.2 and 5.8 Å. More preferably, the values of the depth of the largest groove are between 9.0 and 9.2 Å and the values of the width of the smallest groove are between 5.4 and 5.7Å.

Preferably, the melting temperature is between 55 and 75 ° C (degrees Celsius). More preferably, the melting temperature is between 55 and 62 ° C.

The DNA-binding protein whose values can be calculated by the method is generally a transcription factor, preferably a polyQpolyP domain or a transcription factor selected from the group consisting of SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C / EBP, CREBP1, FOX, Freac7, HFH1, HNF3alfa, Nkx25, POU3F2; Pit1, TTF1, XFD1, AR, C / EBPgamma, Cdc5, FOXD3, HFH3, HNF3beta, MRF2, Oct1, POU6F1, V $ MTATA_B, XFD2, Bach2, CDP CR3, Cdx2, FOXJ2, HFL, HP1, Myc, Myc, PB , TEF, VBP, XFD3, Bm2, COMP1, Evil, FOXP3, GATA4, HFN1, Lhx3, NKX3A, POU1 F1, Pax6, TFIIA or a combination of two or more of these transcription factors.

However, one of skill in the art could determine other classes of transcription factors in order to carry out the method according to the present invention.

In the case where the SMAR Scan® program is applied to perform, for example, large-scale analysis, then, preferably, the aforementioned method further comprises at least one filter that predicts DNA binding sites for the factors of DNA transcription in order to reduce stones.

The principle of this method combines the SMAR Scan® program to calculate the structural characteristics as described before and a filter, such as, for example, the pfsearch program, (from the pftools package described by Bucher P, Karplus K, Moeri N, and Hofmann K, "A flexible search technique based on generalized profiles", Computers and Chemistry, 20: 324, 1996) to predict the binding of some transcription factors.

Examples of filters include, but are not limited to, the pfsearch, MatInspector, RMatch Professional, and TRANSFAC Professional programs.

This combined method uses the structural features of the SMAR Scan® program and the predicted binding of filter-specific transcription factors that can be applied sequentially in any order to select MARs, therefore, depending on the filter, is applied at the beginning or end of the method.

The first level selects sequences from the primary input sequence, and the second level, which consists of the filter, can be used to restrict among the selected sequences those that meet the criteria used by the filter.

In this combined method the filter detects clusters of DNA binding sites using profiles or weighted matrices from, for example, the MatInspector program (Quandt K, Frech K, Karas H, Wingender E, Werner T, "MatInd and MatInspector New fast and versatile tools for detection of consensus matches in nucleotide sequence data ", Nucleic Acids Research, 23, 4878-4884, 1995). The filter can also detect densities of clusters of DNA binding sites.

The combined method is actually a "wrapper" written in Perl for the SMAR Scan @ program and, in the case where the pfsearch program is used as a filter, starting with the pftools package. The combined method performs a two-level treatment using one of these tools (the SMAR Scan @ program or filter) as a potential "filter" at each level, each filter being optional and can be used to calculate the predicted characteristics without filtering.

If the SMAR Scan® program at the first level is used to filter sub-sequences, it must be used with the "all contiguous matches" mode in order for the sequences to appear. If the pfsearch program is used at the first level as the first filter, it must be used only with one profile and a nucleotide distance must be provided. This distance is used to group the matches of the pfsearch program that are located at a distance less than the distance provided for the sequences to appear. The combined method starts the pfsearch program, parses its output and the sequences corresponding to the matches of the pfsearch program appear, which are grouped according to the distance provided. Then, whatever the tool used in the first level, the length of the subsequences thus selected can be systematically extended at both ends according to a parameter called "result extension".

The second and optional level can be used to filter out the sequences (already filtered sequences or unfiltered input sequences) or obtain the results by the SMAR Scan® and / or pfsearch programs without filtering these sequences. If used to filter the second level of the combined method, for each criterion it is necessary to provide the considered exclusion values (result per nucleotide) to filter-separate those sequences (see Fig. 20).

Another object of the present disclosure is also to provide a method for identifying a MAR sequence comprising at least one filter that detects clusters of DNA binding sites using weighted profiles or matrices. Preferably, this method comprises two levels of filters and in this case, the SMAR Scan® program is totally absent from said method. Generally, both levels use the pfsearch program.

Also included in the present disclosure is an identifiable isolated and purified MAR DNA sequence according to the method for identifying a MAR sequence using the disclosed bioinformatics tool, the combined method, or the method comprising at least one filter.

Analysis by the combined method of the entire human genome provided a total of 1,757 putative MARs representing a total of 1,065,305 base pairs. In order to reduce the number of results, a dinucleotide analysis was performed in these 1757 MAR, calculating each of the 16 possible percentages of dinucleotides for each sequence, considering both chains in the 5 'to 3' direction.

Surprisingly, the inventors have shown that all "super" MARs detected by the combined method contain at least 10% of the TA dinucleotide in a contiguous 100 base pair span. Preferably these sequences contain at least 33% of the TA dinucleotide over a 100 base pair span.

The inventors have also shown that these same sequences further contain at least 12% of the AT dinucleotide in a contiguous 100 base pair span. Preferably they contain at least 33% of the AT dinucleotide in a contiguous 100 base pair span.

Another aspect of the description is to provide a purified and isolated MAR DNA sequence from any of the MAR described above, comprising a sequence selected from sequences SEQ ID No. 1 to 27, one of its complementary sequences, one of its parts that it shares at least a 70% nucleotide length, one of its molecular chimeras, and one of its combinations and variants.

Preferably, said isolated and purified MAR DNA sequence comprises a sequence selected from sequences SEQ ID No. 24 to 27, one of its complementary sequences, one of its parts that shares at least a nucleotide length of 70%, one of its molecular chimeras and one of its combinations and variants. These sequences 24 to 27 correspond to those detected by the combined method and show a higher activity that increases protein production than sequences 1 to 23.

The present disclosure also encompasses the use of an isolated and purified DNA sequence comprising a first nucleotide sequence isolated from a matrix-binding region (MAR) which is a MAR nucleotide sequence selected from the group comprising

-

an isolated and purified DNA sequence according to the invention,

-

the sequence SEQ ID No 27, or one of its complementary sequences,

to increase protein production activity in a eukaryotic host cell.

Said isolated and purified DNA sequence generally further comprises one or more regulatory sequences, known in the art, for example a promoter and / or an enhancer, polyadenylation sites and splice junctions, generally employed for the expression of the protein or may optionally encode a selectable marker. Preferably, said isolated and purified DNA sequence comprises a promoter that is operably linked to a gene of interest.

The DNA sequences of this invention can be isolated according to standard PCR protocols and methods well known in the art.

Promoters that can be used, provided that they are compatible with the host cell, are, for example, promoters obtained from the genomes of viruses such as polyomaviruses, adenoviruses (such as Adenovirus 2), papillomaviruses (such as bovine papillomaviruses), viruses from avian sarcoma, cytomegalovirus (such as the immediate early promoter of human or murine cytomegalovirus), a retrovirus, hepatitis B virus and simian virus 40 (such as early and late promoters of SV 40) or promoters obtained from promoters mammalian heterologists, such as the actin promoter or an immunoglobulin promoter or heat shock promoters. Such regulatory sequences direct constitutive expression.

Furthermore, the isolated and purified DNA sequence could comprise regulatory sequences that are capable of directing the expression of nucleic acids preferably in a particular cell type (eg, tissue-specific regulatory elements are used to express nucleic acid). Tissue specific regulatory elements are known in the art. Suitable non-limiting examples of tissue specific promoters include the albumin promoter (liver specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymph tissue specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular T cell receptor promoters (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729 -740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (eg, the neurofilament promoter; Byrne and Ruddle, 1989. Proc.Natl. Acad. Sci. USA 86: 5473- 5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916) and mammary gland-specific promoters (eg, milk serum promoter; US Patent No.

4,873,316 and European Patent Application No. 264,166).

Development-regulated promoters are also included. Examples of such promoters include, for example, promoters of murine homeotic genes (Kessel and Gruss, 1990. Science 249: 374-379) and alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537. -546).

Regulable gene expression promoters are well known in the art and include, as a non-limiting example, any promoter that modulates the expression of a gene encoding a desired protein by binding an exogenous molecule, such as the CRE / LOX system, the TET system, doxycycline system, NFkappaB / UV light system, Leu3p / isopropyl malate system and GLVPc / GAL4 system (see for example, Sauer, 1998, Methods 14 (4): 381-92; Lewandoski, 2001, Nat Rev. Genet 2 (10): 743-55; Legrand-Poels et al., 1998, J. Photochem. Photobiol. B. 45:18; Guo et al., 1996, FEBS Lett. 390 (2): 191 -5; Wang et al., PNAS USA, 1999, 96 (15): 84838). However, one of skill in the art could determine other classes of promoters that are suitable for carrying out the present invention.

Enhancers may then be optionally included in the purified DNA sequence of the invention which will then belong to the regulatory sequence, for example the promoter.

The "gene of interest" or "transgene" preferably encodes a protein (structural or regulatory protein). As used herein "protein" generally refers to peptides and polypeptides that have more than about ten amino acids. Proteins can be "homologous" to the host (ie, endogenous to the host cell to be used) or "heterologous" (ie, foreign to the host cell to be used), such as a human protein produced by yeast. Protein can be produced as an insoluble aggregate or as a soluble protein in the periplasmic space or cytoplasm of the cell or in the extracellular medium.Examples of proteins include hormones, such as growth hormone or erythropoietin (EPO), growth factors, such such as epidermal growth factor, analgesic substances such as enkephalins, enzymes such as chymotrypsin, hormone receptors or growth factors, antibodies and also include pr Oteins generally used as a display marker, for example the green fluorescent protein.

Preferably, the purified DNA sequence further comprises at least a second nucleotide sequence of an isolated matrix binding region (MAR) selected from the group comprising:

-

an isolated and purified DNA sequence according to the invention,

-

sequence SEQ ID No. 27, or one of its complementary sequences,

to increase protein production activity in a eukaryotic host cell.

The isolated matrix-binding region (MAR) nucleotide sequences could be identical or different. Alternatively, a first and a second identical MAR nucleotide sequence are used.

Preferably, the MAR nucleotide sequences are located at both the 5 'and 3' ends of the sequence containing the promoter and the gene of interest. But the invention also provides for the fact that said first and / or at least second MAR nucleotide sequences are located in a different sequence from that containing the promoter and the gene of interest.

Also included within the scope of this disclosure is the isolated and purified DNA sequence comprising a first isolated matrix binding region (MAR) nucleotide sequence which is a MAR nucleotide sequence selected from the group comprising :

-

an isolated and purified DNA sequence that has activity that increases protein production,

-

an identifiable isolated and purified MAR DNA sequence according to the method for identifying a MAR sequence using the described bioinformatics tool, the combined method or the method comprising at least one described filter,

-

sequences SEQ ID No. 1 to 27,

-

an isolated and purified cLysMAR element and / or fragment,

-

a synthetic MAR sequence comprising elements and / or fragments of natural MAR assembled between connecting sequences,

one of its complementary sequences, one of its parts that share a nucleotide length of at least 70%, one of its molecular chimeras, one of its combinations and variants that can be used to increase protein production activity in a cell eukaryotic host by introducing the isolated and purified DNA sequence into a eukaryotic host cell according to well-known protocols. The generally applied methods of introducing DNA into eukaryotic host cells are, for example, direct introduction of cloned DNA by microinjection or microparticle bombardment; electrotransfer; use of viral vectors; encapsulation in a carrier system; and use of transfection reagents, such as calcium phosphate, diethylaminoethyl (DEAE) -dextran, or commercial transfection systems such as Lipofect-AMINE 2000 (Invitrogen). Preferably, the transfection method used to introduce the purified DNA sequence into a eukaryotic host cell is the method of transfecting a eukaryotic cell as described below.

The isolated and purified DNA sequence can be used in the form of a circular vector. Preferably, the isolated and purified DNA sequence is used in the form of a linear DNA sequence as a vector.

As used herein, "plasmid" and "vector" are interchangeable, although plasmid is the most commonly used form of vector. However, the invention is understood to include other forms of expression vectors, such as, but not limited to, viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions.

The present invention further includes a method for transfection of a eukaryotic host cell, said method comprising:

a) introducing into said eukaryotic host cell at least one purified DNA sequence comprising at least one DNA sequence consisting of a MAR sequence according to the invention,

b) subjecting in a defined time said eukaryotic host cell to at least one additional transfection step with at least one purified DNA sequence comprising at least one DNA sequence of interest and / or with at least one isolated and purified DNA sequence comprising a nucleotide sequence of MAR or other chromatin modifying elements and

c) selecting said eukaryotic host cell.

Preferably, in step b) at least two to four transfection steps are applied.

In order to select successfully transfected cells, a gene encoding a selectable marker (eg, antibiotic resistance) is generally introduced into the host cells along with the gene of interest. The gene encoding a selectable marker could be located in the purified DNA sequence comprising at least one DNA sequence of interest and / or at least one isolated and purified DNA sequence consisting of a MAR nucleotide sequence or other elements Chromatin modifiers or optionally could be co-introduced separately, eg into a plasmid. Several selectable markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. The amount of drug can be adapted as desired, in order to increase productivity.

Generally, one or more selectable markers are used. Preferably, the selectable markers used in each different transfection step are different. This allows transformed cells to be selected that are "multi-transformed" using, for example, two different antibiotic selections.

Any eukaryotic host cell capable of protein production and lacking a cell wall can be used in the methods of this invention. Examples of useful mammalian host cell lines include human cells, such as the human embryonic renal cell line, 293 cells, or 293 cells sub-cloned for suspension culture, Graham et al., J. Gen Virol. 36, 59 (1977)), human cervical carcinoma cells, such as (HELA, ATCC CCL 2), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065); rodent cells, such as hamster pup kidney cells (BHK, ATCC CCL 10), Chinese hamster / -DHFR ovary cells (CHO, Urlaub and Chasin, Proc. Nad. Acad. Sci. USA, 77, 4216 (1980)), mouse Sertoli type cells (TM4, Mather, Biol. Reprod., 23, 243-251 (1980)), mouse mammary tumor cells (MMT 060562, ATCC CCL 51); and cells from other mammals, such as the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); and myeloma cells (eg, NS0) / hybridoma.

Preferably, the selected transformed eukaryotic host cells are highly protein-producing cells with a production rate of at least 10 pg per cell per day.

Most preferred for human use in the present invention are CHO cells.

The DNA sequence of interest from the isolated and purified DNA sequence is usually a gene of interest that preferably encodes a protein operably linked to a promoter as described above. The isolated and purified DNA sequence comprising at least one DNA sequence of interest could further comprise the DNA sequence of interest of the MAR nucleotide sequence or other chromatin modifying elements.

The isolated and purified DNA sequence comprising a MAR nucleotide sequence is selected, for example, from the group comprising sequences SEQ ID No. 1 to 27 and / or particular elements of cLysMAR, for example, regions B, K and F, as well as fragments and elements and their combinations, as described above. Other chromatin modifying elements are, for example, border elements (BE), locus control regions (CSF), and ubiquitous chromatin-opening elements (UCOE) (see Zahn-Zabal et al., Already cited). An example of multiple host cell transfections is shown in Example 12 (Table 3). The first transfecting step (primary transfection) is performed with the gene of interest (SV40EGFP) alone, with a MAR nucleotide sequence alone, or with the gene of interest and a MAR nucleotide sequence (MARSV40EGFP). The second transfecting step (secondary transfection) is carried out with the gene of interest (SV40EGFP) alone, with a nucleotide sequence of MAR alone or with the gene of interest and a nucleotide sequence of MAR (MAR-SV40EGFP), in all possible combinations resulting from the first transfectant stage.

Preferably, the eukaryotic host cell is transfected:

a) introducing a purified DNA sequence comprising a DNA sequence of interest and additionally a MAR nucleotide sequence, and

b) subjecting in a defined time said transfected eukaryotic host cell to at least one step of

further transfection with the same purified DNA sequence comprising a DNA sequence from

of interest and additionally a MAR nucleotide sequence from step a).

Also preferably, the MAR nucleotide sequence of the isolated and purified DNA sequence is selected from the group comprising:

-

an isolated and purified DNA sequence that has activity that increases protein production,

-

an isolated and purified DNA sequence identifiable from MAR according to the method of identifying a MAR sequence using the described bioinformatics tool, the combined method or the method comprising at least one filter,

-

sequences SEQ ID No. 1 to 27,

-

an isolated and purified cLysMAR element and / or fragment,

-

a synthetic MAR sequence comprising a natural MAR element and / or fragments assembled between connecting sequences,

one of its complementary sequences, one of its parts that shares at least a nucleotide length of 70%, one of its molecular chimeras, one of its combinations and variants.

Surprisingly, a synergy has been observed between the first and second transfection. A particular synergy has been observed when the elements of the MAR are present in one or both stages of transfection. Multiple transfections of cells have been carried out with pMAR alone or in combination with various expression plasmids, using the method described above. For example, Table 3 shows that transfecting cells twice with the plasmid pMAR-SV40EGFP gave the highest expression of GFP and the highest degree of potentiation of all conditions (4.3 times). In contrast, transfecting the vector twice without the MAR gave little or no enhancement, 2.8 times, rather than the expected double increase. This demonstrates that the presence of the MAR elements at each transfection stage is of particular interest to achieve maximum protein synthesis.

As a particular example of the transfection method, said purified DNA sequence comprising at least one DNA sequence of interest can be introduced in the form of multiple unbound plasmids, comprising a gene of interest operably linked to a promoter, a selectable marker gene and / or elements that increase protein production, such as MAR sequences.

The relationship between the former and subsequent DNA sequences can be tailored as required for the use of specific cell types and can be obtained by those skilled in the art by routine experimentation.

The time defined for further transformations of the transformed primary cells closely depends on the cell cycle and its duration. Usually, the defined time corresponds to intervals related to the cell division cycle.

Therefore, this precise time can be adapted as required for the use of specific cell types, and can be obtained by those skilled in the art by routine experimentation.

Preferably, the defined time is the time at which the host cell has just entered the same phase of a second or subsequent cycle of cell division, preferably the second cycle.

This time is usually between 6 hours and 48 hours, preferably between 20 hours and 24 hours after the previous transfection event.

Also encompassed by the present invention is a method of transfecting a eukaryotic host cell, said method comprising co-transfecting into said eukaryotic host cell at least a first isolated and purified DNA sequence comprising at least one DNA sequence of interest and a second isolated and purified DNA sequence comprising at least one MAR nucleotide sequence selected from the group comprising: an isolated and purified DNA sequence according to the invention, the sequence SEQ ID No. 27 or one of its complementary sequences.

A process for the production of a protein is also provided where a eukaryotic host cell is transfected according to the transfection methods defined in the present invention and grown in a culture medium under conditions suitable for the expression of the protein. Said protein is finally recovered according to any recovery process known to those skilled in the art.

As an example the following process could be used for protein production. The eukaryotic host cell transfected by the transfection method of the present invention is used in a process for the production of a protein by culturing said cell under conditions suitable for the expression of said protein and recovering said protein. Suitable culture conditions are those commonly used for in vitro culture of eukaryotic cells as described, for example, in WO 96/39488. The protein can be isolated from cell culture by conventional separation techniques, such as, for example, fractionation on immunoaffinity or ion exchange columns; precipitation; Reverse phase HPLC; chromatography; chromato focus; SDS-PAGE; gel filtration. One of skill in the art will appreciate that suitable purification methods for the polypeptide of interest may require modifications to account for changes in polypeptide characteristics in expression in recombinant cell culture.

The functionality of the proteins that are produced according to this invention can be analyzed by a variety of methods. For example, the presence of antigenic epitopes and the ability of proteins to bind ligands can be determined by Western blot assays, fluorescent cell sorting assays, immunoprecipitation, immunochemical assays, and / or competitive binding assays, as well as any other assay that measure specific binding activity.

The proteins of this invention can be used in a number of practical applications including, but not limited to:

one.

Immunization with recombinant host protein antigen as a virus / pathogen antagonist.

2.

Production of membrane proteins for diagnostic or screening tests.

3.

Membrane protein production for biochemical studies.

Four.

Production of membrane proteins for structural studies.

5.

Antigen production for the generation of antibodies for immuno-histochemical mapping, including mapping of orphan receptors and ion channels.

The present invention also provides a host eukaryotic cell transfected according to the preceding transfection methods. Preferably, the host eukaryotic cell is a non-human host cell line.

As already described, examples of useful mammalian host eukaryotic cell lines include human cells, such as human embryonic renal cell line (293 cells or 293 cells subcloned for suspension culture, Graham et al., J. Gen Virol. 36, 59 (1977)), human cervical carcinoma cells, such as (HELA, ATCC CCL 2), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065); rodent cells, such as baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster / -DHFR ovary cells (CHO, Urlaub and Chasin, Proc. Nad. Acad. Sci. USA, 77, 4216 (1980)), mouse Sertoli type cells (TM4, Mather, Biol. Reprod., 23, 243-251 (1980)), mouse mammary tumor cells (MMT 060562, ATCC CCL 51); and cells from other mammals, such as the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); and myeloma cells (eg, NS0) / hybridoma.

Most preferred for uses in the present invention are Chinese Hamster Ovary (CHO) cells.

The present invention also provides a mixture or kit for cell transfection comprising at least one isolated and purified DNA sequence according to the invention.

The invention further comprises a transgenic non-human organism characterized in that at least some of its cells have stably incorporated at least one DNA sequence according to the invention.

Preferably some of the cells of these transgenic organisms have been transfected according to the methods described herein.

A transgenic non-human organism characterized in that its genome has stably incorporated at least one DNA sequence according to the invention is also considered in the present invention.

Transgenic eukaryotic organisms that may be useful for the present invention are selected for example from the group comprising mammals (monkey, mouse, etc.) and in particular laboratory animals, such as rodents in general, insects (Drosophila, etc.), fish (zebrafish, etc.), amphibians (frog, newt, etc.) and other simpler organisms, such as the C. elegans worm, yeast, etc ...

It is still another object of the present disclosure to provide a computer readable medium comprising computer executable instructions for performing the method of identifying a MAR sequence as described in the present disclosure.

The foregoing description will be more fully understood with reference to the following Examples. However, such Examples are illustrative of methods of practicing the present invention and are not intended to limit its scope.

EXAMPLES

Example 1: SMAR Scan® program and MAR sequences

A first rough evaluation was performed with the SMAR Scan® software, analyzing experimentally defined human MARs of MAR and non-MAR sequences. As MAR sequences, the previous results of the analysis of human MAR from the SMARt DB were used to graphically represent a histogram of densities for each criterion, as shown in Fig. 1. Similarly, the sequences were also analyzed and graphically represented. that are not from MAR. As non-MAR sequences, all RefSeq contigs (contiguous clones) from chromosome 22 were used, considering that the latter was large enough to contain a negligible part of MAR sequences compared to the part of non-MAR sequences .

The density distributions shown in Fig. 1 are all skewed with a long tail. For maximum curvature, maximum depth of major groove, and maximum width of minor groove, distributions are skewed to the right. For the minimum melting temperature, the distributions are skewed to the left, which is natural given the inverse correspondence of this criterion with the other three. For MAR sequences, biphasic distributions with a second weak peak are really evident. And between the distributions of MAR sequences and non-MAR sequences, a clear shift is also visible on each graph.

Among all the human MAR sequences used, on average only about 70% of them have a higher value than the 75th quantile of distribution of human MARs, and this for the four different criteria. Similarly, relative to the second weak peak of each human MAR distribution, only 15% of human MAR sequences are responsible for these peripheral values. Among this 15% of human MAR sequences, the majority are very well documented MAR sequences, used to isolate a transgene from positional effects, such as MAR of the interferon locus, MAR of the beta-globin locus (Ramezani A, Hawley TS, Hawley RG, "Performance-and safety-enhanced lentiviral vectors containing the human interferon-beta scaffold attachment region and the chicken beta-globin insulator", Blood, 101: 4717-4724, 2003), or the MAR of apolipoprotein (Namciu, S, Blochinger KB, Foumier REK, "Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster", Mol. Cell Biol., 18: 2382-2391, 1998).

Always with the same data, human MAR sequences were also used to determine the association between the four calculated theoretical structural properties and the AT content. Fig. 2 represents the scatterplot and the corresponding correlation coefficient r for each pair of criteria.

Example 2: MAR Sequence Distribution Charts by Organism

As previously explained, MAR sequences from the SMARt DB from other organisms were also recovered and similarly analyzed. The MAR sequence density distributions for mouse, chicken, bicolor sorghum and man are plotted and plotted in Fig. 3.

Example 3: Prediction of the MAR of the complete chromosome 22

All RefSeq contigs on chromosome 22 were analyzed by the SMAR Scan® program this time using the default settings. The result is that the SMAR Scan® program predicted a total of 803 MAR, with an average length of 446 bp, which means an average of a predicted MAR of 42,777 bp. The total length of the predicted MARs corresponds to 1% of the length of chromosome 22. The AT content of the predicted regions ranged from 65.1% to 93.3%; the mean AT content of all these regions being 73.5%. Therefore, the predicted MARs were rich in AT, while chromosome 22 is not rich in AT (52.1% AT).

The SMARTest program was also used to analyze the complete chromosome 22 and 1387 candidates for MAR were obtained, with an average length of 494 bp, which represents an average of a MAR predicted by 4,765 bp. The total length of the predicted MARs corresponds to 2% of chromosome 22. Among all the MARs predicted by the two computer programs, 154 predicted MARs were found by both programs, representing respectively 19% and 11% of the MARs predicted by the SMAR Scan® and SMARTest programs. Given the mean length of the MARs predicted by the SMAR Scan® and SMARTest programs, the probability of having an overlap by chance between the predictions of the SMAR Scan® and SMARTest programs is 0.0027% per prediction.

To assess the specificity of the predictions by the SMAR Scan® program, analyzes were performed by the SMAR Scan® program on randomly shuffled sequences of chromosome 22 (Fig. 4). Shuffled sequences were generated using 4 different methods: by cleavage of chromosome 22 into 10 bp non-overlapping windows and shuffling the nucleotides in each window separately; for "scrambling" which means a permutation of all nucleotides on the chromosome; by "rubbling" which means a segmentation of the chromosome into fragments of 10 bp and a random assembly of these fragments, and finally by Markov chains of order 1, the different states being the totality of the different DNA dinucleotides and based on probabilities Transition between these states on chromosome 22 scanning. For each shuffling method, five shuffled 22 chromosomes were generated and analyzed by the SMAR Scan® program. Regarding the number of matches, an average of 3,519,170 matches (standard deviation: 18,353) were found for chromosome 22 permuted within the non-overlapping windows of 10 bp, 171,936.4 matches (standard deviation: 2,859.04) for the sequences subjected to scambling and 24,708.2 matches (standard deviation: 1,191.59) for chromosome 22 subjected to rubbling and 2,282 mean matches (standard deviation: 334.7) for the chromosomes generated according to the Markov chain models of order 1 of chromosome 22, which represents respectively 185% (standard deviation: 0.5% of the mean), 9% (standard deviation: 1.5%), 1% (standard deviation: 5 %) and 0.1% (standard deviation: 15%) of the number of matches found with natural chromosome 22. For the number of predicted MARs, which therefore means contiguous coincidences of length greater than 300 bp, 1,997 MARs with chromosome 22 shuffled within the 10 bp windows were predicted (standard deviation: 31., 2), and were found only 2.4 MAR candidates in sequences subjected to scrambling (standard deviation: 0.96) and none for sequences subjected to rubbling and for sequences generated according to the Markov chain model, which represents respectively 249% and less than 0.3% of the number of predicted MARs found with the natural chromosome 22. These data provide indications that the SMAR Scan @ program detects specific DNA elements whose organization is lost when shuffling DNA sequences.

Example 4: Analysis of known matrix binding regions at the interferon locus with the SMAR Scan® program

The relevance of MAR prediction by the SMAR Scan® program was investigated by analyzing recently published MARs from the human interferon gene cluster on the short arm of chromosome 9 (9p22). Goetze et al. (Already cited) described a comprehensive analysis of the WP18A10A7 locus to analyze the suspected correlation between BURs (referred to in this case as stress-induced duplex destabilization or SIDD) and in vitro binding to the nuclear matrix (Fig. 9, bottom). Three of the SIDD peaks were in agreement with the in vitro binding assay, while others did not match the matrix binding sites. Inspection of the interferon locus with the SMAR Scan® program (Fig. 9, top) indicated that three major peaks accompanied by clusters of regulatory binding sites SATB1, NMP4 and MEF2 were well correlated with active MARs. Therefore, the inventors concluded that the existence of predicted CUEs and binding sites for these transcription factors is not restricted to cLysMARs, but may be a general property of all MARs. These results also imply that the SMAR Scan® program efficiently detects MAR elements from genomic sequences.

Example 5: Prediction accuracy by SMAR Scan® program and comparison with other prediction tools

The accuracy of the SMAR Scan® program was evaluated using six genomic sequences for which experimentally determined MARs had been mapped. In order to make a comparison with 5 other prediction tools, the sequences analyzed are the same as the sequences previously used to compare the MAR-Finder and SMARTest programs. These genomic sequences are three plant sequences and three human sequences (Table 1) totaling 310,151 bp and 37 MAR experimentally defined. The results of the SMARTest and MAR-Finder programs in Table 1 come from a previous comparison (Frisch M, Frech K, Klingenhoff A, Cartharius K, Liebich I and Werner T, "In silico prediction of scaffold / matrix attachment regions in large

10 genomic sequences ", Genome Research, 12: 349-354, 2001). The MAR-Finder program has been used with the default parameters except for the threshold value that is set at 0.4 and for the protamine locus analysis , the rule of AT richness has been excluded (to detect MARs not rich in AT as was done for the protamine locus).

Table 1: SMAR Scan® Program Accuracy Assessment

Description and reference of the sequences

Length (kb) Experimentally defined MAR positions (kb) SMARTest Predicted Positions (kb) MAR-Finder Predicted Positions (kb) SMAR Scan Predicted Positions (kb)

SH2 gene assumption of the sub-unit

30,034  0.0-1.2 - - -

ADP-glusosapirofosphorylase and putative A1 reductase dependent gene

5.4-7.4 6.5-7.0 152-15.7 162-16.6 -15.7-15.9 - -15.6-16 -

NADPH by Oryza Sativa

17.3-18.5 17.6-18.3 17.5-18.4 17.6-18.2

(U700541) [4]

20.0-23.1 19.6-20.1 20.1-21.3 23.6-23.9 26.0-25.4 27.5-27.9 19.8-20.4 21.3-21.5 23.9-24.2 24.7-25.1 - 21.6-22 -23.4-23.8 -

ADP Subunit SH2 Genes

42,446  0.0-1.6 - - -

glucosapirofosforilasa and reductasa dependent A1-b

7.1-9.7 -21.3-21.9 - 7.4-7.7 21.5-21.8

NADPH of sorghum bicolor (AF010283) [4]

22.4-24.7 22.9-24.0 -27.3-27.6 23.2-24.2 -26.9-27.6 22.9-23.2 23.6-24.0 27.3-27.6

32.5-33.7

- - 33.4-33.9

41.6-42.3

- - -

Clone 110K5

78,195  ~ 0.9 - - -

BAC of

bicolor sorghum

~ 5.8 - - -

 (AF124045) [37]

~ 6.3 - - -

~ 9.3

- - -

~ 15.0

15.1-15.8 - -

~ 18.5

- - -

~ 21.9

29.7-22.0 - 21.4-21.9

~ 23.3

- - -

~ 25.6

- - -

~ 29.1

- - 29.2-29.5

~ 34.6

- - -

Table 1 (Continuation)

Description and reference of the sequences

Length (kb) Experimentally defined MAR positions (kb) SMARTest Predicted Positions (kb) MAR-Finder Predicted Positions (kb) SMAR Scan Predicted Positions (kb)

-

-

39.0-40.0

~ 44.1

44.1-44.5 -

~ 48.5

47.9-49.5 - 47.9-49.4 - 48.1-48.6 48.8-49.3

~ 57.9

- - -

~ 62.9

63.1-63.7 - -

~ 67.1

- - -

~ 69.3

- - -

~ 73.7

74.3-74.7 - 74.3-74.6

Alpha-1-antitrypsin intergenic region

30,461  2.6-6.3 5.5-6.0 3.0-3.2 5.4-5.8

human and globulin binding to

- 5.1-6.0 -

corticosteroids

22.0-30.4 26.7-2.2 24.9-25.3 25.8-26.4

(AF156545), [35]

27.5-27.8 25.5-25.8

-

26.2-26.4

-

-

27.5-29.2

-

Protamine locus

53,060  8.8-9.7 - 8.0-8.9 -

human

(U15422) [24]

32.6-33.6 - 33.9-34.8 * -

37.2-39.4

- 33.9-34.8 * -

51.8-53.0

- - * -

Human beta-globin locus

75,955  1.5-3.0 - - 2.3-2.6

(U01317), [21]

15.6-19.0 18.0-18.4 -34.4-34.9 15.5-16.0 18.0-18.4 - 15.3-15.6 -

44.7-52.7

- 50.6-50.8 -

60.0-70.0

55.6-57.1 59.8-60.3 65.6-66.0 67.6-67.9 68.8-69.1 56.5-57.2 58.1-58.5 63.0-63.6 69.7-69.3 - -62.5-63.1 -66.3-66.7 -

Sum (kb)

310,151 at least 56.1 14.5 13.8 9.5

Total numbers:

37 28 25 22

Average kb / MAR predicted

11,076 12,406 14,097

True positives [number of experimentally defined MARs found]

19 [14] 20 [12] 17 [14]

false positives

9 5 5

false negatives

2. 3 25 2. 3

Specificity

19/28 = 68% 20/25 = 80% 17/22 = 77%

Sensitivity

14/37 = 38% 12/37 = 32% 14/37 = 38%

Six different genomic sequences, three vegetable and three human sequences, for which experimentally defined MARs are known, were analyzed with the MAR-Finder, SMARTest and SMAR Scan® programs. True positive matches appear in the table above in bold numbers, the minus sign (-) indicates 5 false negative matches. Some of the longest experimentally defined MARs contained more than one in silico prediction (that is, made by the program), each of which was counted as a true positive match. Therefore, the number of true predictions in silico is greater than the number of experimentally defined MARs found. Specificity is defined as the ratio of true positive predictions to total number, while sensitivity is defined as the ratio of experimentally defined ADRs.

10 found and the total number. * The wealth rule in AT excluded the use of the MAR-Finder program.

The SMARTest program predicted 28 regions as MAR, 19 (true positive) of these are correlated with experimentally defined MAR (specificity: 68%), while 9 (32%) are located in non-MAR (false positive) regions. Since some of the longest experimentally determined MARs contain more than one in silico prediction, the 19 true positives actually correspond to 14 different experimentally defined MARs (sensitivity: 38%). The MARFinder program predicted 25 regions as MAR, 20 (specificity: 80%) of these are correlated with experimentally defined MARs corresponding to 12 different experimentally defined MARs (sensitivity: 32%). The SMAR Scan @ program predicted 22 regions, with 17 true positives (specificity: 77%) that coincide with 14 different experimentally defined MARs (sensitivity: 38%).

As another example, the same analysis has been applied to human chromosomes 1 and 2 and led to the determination of 23 MAR sequences (SEQ ID No. 1 to 23). These sequences are included in Annex 1 in ST25 format.

Example 6: Analysis of the entire genome using the combined method (SMAR Scan®-pfsearch)

In order to analyze the potential correlation between the structural characteristics analyzed by the SMAR Scan @ program and the functional activity of the S / MAR, the complete human genome has been analyzed with the combined method with very restrictive parameters, in order to obtain sequences with the highest values for the theoretical structural characteristics analyzed by computer, which hereafter are called "super" S / MAR. This was done in the hope of obtaining predicted MAR elements with a high potential to increase transgene expression and recombinant protein production. The supposed S / MAR thus obtained were first analyzed from the perspective of bioinformatics in an attempt to characterize and classify them.

6.1 S / MAR predicted by analysis of the complete human genome

As a complete human genome sequence, all human contigs from RefSeq (National Center for Biotechnology Information, The NCBI Handbook [Internet]. Bethesda (MD): National Library of Medicine (US), Oct. Chapter 17, The Reference Sequence) were used. (RefSeq) Project, 2002 (Available at http://www.ncbi.nih.gov/entrez /query.fcgi?db=Books) (publication 5) and analyzed with the combined method, using the SMAR Scan® program as filter in the first level processing, using the default settings except for the maximum curvature exclusion value, while a restricted threshold of 4.0 degrees (instead of 3.202 degrees) has been used for the DNA curvature criterion.

In the second level processing, the transcription factor binding sites in the selected sequences of the previous step were searched without filtering these sequences.

Analysis by the combined method of the complete human genome gave a total of 1,757 putative "super" S / MARs representing a total of 1,065,305 bp (0.35% of the complete human genome). Table 2 shows for each chromosome: its size, its number of genes, its number of predicted S / MAR, its density of S / MAR for each gene and its kb per S / MAR. This Table shows that there are very different gene densities for each predicted S / MAR for the different chromosomes (the standard deviation represents more than 50% of the mean predicted S / MAR gene density and the difference between the gene density higher and lower by S / MAR is 6.5 times). Table 2 also shows that the kb by S / MAR varies less than the gene density by S / MAR (the standard deviation represents 25% of the mean kb by S / MAR and the difference between the largest and smallest kb by S / MAR is 3.2 times).

Table 2: Number of predicted S / MAR by chromosome. The number of genes per chromosome corresponds to NCBI human genome statistics (Build 34, Version 3)

Chromosome

 Number of genes per chromosome Chromosome size (million bp) Predicted S / MAR number S / MAR gene density Kb by S / MAR

one

2544 230 85 29.9 2705

2

1772 241 143 12.3 1685

3

1406 198 101 13.9 1960

4

1036 190 118 8.7 1610

5

1233 180 116 10.6 1551

6

1247 170 94 13.2 1808

7

1383 160 179 7.7 1754

8

942 145 77 12.2 1883

9

1100 119 48 22.9 2479

10

1003 133 71 14.1 1873

eleven

1692 132 67 25.2 1970

12

1278 131 78 16.3 1679

13

506 97 70 7.2 1385

14

1168 88 36 32.4 2444

fifteen

895 83 35 25.5 2371

16

1107 81 41 27 1975

17

1421 80 37 38.4 2162

18

396 75 31 7.7 1470

19

1621 56 36 45.02 1555

twenty

724 60 28 25.8 2142

twenty-one

355 3. 4 18 19.7 1888

22

707 3. 4 28 25.2 1214

X

1168 154 170 6.8 905

AND

251 25 30 83 833

Sum Mean Standard Deviation

26,955 1,123 510 3,050 127 72.8 1,757 73 45 457 19 10 43,312 1,804 462

Data from the National Center for Biotechnology Information, The NCBI Handbook [Internet]. Bethesda (MD): National Library of Medicine (US), Oct. Chapter 17, The Reference Sequence (RefSeq) Project, 2002 (Available at http:

5 //www.ncbi.nih.gov/entrez/query.fcgi?db=Books) are based on GenBank annotations. Chromosome sizes are the sum of the corresponding lengths of human contigs from RefSeq (National Center for Biotechnology Information, The NCBI handbook [Internet]. Bethesda (MD): National Library of Medicine (US), Oct. Chapter 17, The Reference Sequence (RefSeq) Project, 2002 (Available at http: //www.ncbi.nih.gov/entrez /query.fcgi?db=Books) (publication 5).

10 6.2 Bioinformatic analysis of the "super" MAR for transcription factor binding sites.

The potential sites for transcription factor binding of the 1,757 predicted "super" S / MAR sequences previously obtained by the SMAR Scan @ program were then analyzed. This has been achieved using the RMatch ™ Professional program (Kel AE, Gossling E, Reuter I, Cheremushkin E, KelMargoulis OV, Wingender E, MATCH: "A tool for searching transcription factor binding sites in DNA sequences", Nucleic Acids Res. 31 (13): 35769, 15 2003), a tool based on a weighted matrix, based in turn on the TRANSFAC program (Wingender E, Chen X, Fricke E, Geffers R, Hehl R, Liebich I, Krull M, Matys V , Michael H, Ohnhauser R, Pruss M, Schacherer F, Thiele S, Urbach S, "The TRANSFAC system on gene expression regulation", Nucleic Acids Research, 29 (1): 2813, 2001). The Match 2.0 Professional program has been used with most of the default settings. Analysis by the Match program was based on the TRANSFAC Professional program, version 8.2 (20040630). Sums

20 of all predictions of transcription factor binding sites in the 1757 sequences analyzed according to the Match program are shown in Table 3. Based on this Table, only transcription factors totaling at least 20 matches in the 1757 analyzed sequences they were considered for further analysis.

Given below are some of the human transcription factors that are most frequently predicted

25 to bind to the 1,757 putative S / MAR sequences and their description by the Match program: Cdc5 (protein 5 of cell division control) a transcriptional regulator / repressor, Nkx3A an androgen-regulated homeodomain protein, POU1F1 (factor 1 pituitary-specific positive transcription) that is pituitary-specific and stimulates cell proliferation. Therefore, in the activity of MARs, in addition to SATB1, NMP4 and MEF2, other transcription factors may participate.

Table 3 is a summary of all predictions of transcription factor binding sites (totaling 20 matches or more) in the 1,757 sequences analyzed.

AP1

one AR 2 Bach2 one Brn2 one

C / EBP

twenty  C / EBPgamma 5 CDP CR3 one COMP1 2

CREBP1

3. 4 Cdc5 858 Cdx2 35 Evil 472

FOX

78 FOXD3 79 FOXJ2 244 FOXP3 29

Freac7

272  GATA1 2 GATA3 142 GATA4 125

HFH1

12  HFH3 one HLF 275 HNF1 337

HNF3alpha

2. 3  HNF3beta 71  HP1 2 Lhx3 22

MEF2

114  MRF2 57  Myc 18 NKX3A 849

Nkx25

2 Oct1 191 PBX 5 POU1F1 483

POU3F2

eleven POU6F1 29  Pax3 3 Pax6 twenty

Pit1

505 SRF 8 TEF 2852 TFIIA 14

TTF1

one V $ MTATA_B 4 VBP 53 Vmw65 one

XFD1

65  XFD2 418  XFD3 2

6.3 Bioinformatic analysis of the "super" MARs to determine nucleotide frequencies.

Various computer analyzes were performed in order to easily identify "super" S / MAR sequences

5 using an explicit criterion that could be identified without the use of a computer. Among these, a dinucleotide analysis was performed in the 1757 super MAR, calculating each of the 16 possible percentages of dinucleotides for each sequence considering both chains in the 5 '> 3' direction.

A summary (minimum, maximum, median, mean, 25th percentile and 75th percentile), as well as the histograms of each percentage of dinucleotide in the 1757 S / MAR sequences, are respectively represented in Table 4.

10 performed a similar analysis on randomly selected sequences from the human genome, randomly representing the selected non-S / MAR sequences (which, however, might contain some MARs). Table 5 represents respectively a summary of the analysis of the dinucleotide content of these sequences.

Table 4: Percentages of dinucleotides in 1757 S / MAR sequences

AA%

AC% AG% AT%

Minimum

0.000 0.0000 0.0000 18.50

25th percentile

4,234 0.9372 0.1408 32.11

Median

7,843 2,2408 0.4777 34.68

Half

7,184 3.2117 1.0865 34.32

75th percentile

10,110 4.7718 1,5096 36.94

Maximum

17,290 12.9479 8.1230 50.00

 AC%

DC% CG% CT%

Minimum

0.0000 0.00000 0.0000 0.0000

25th percentile

0.9695 0.00000 0.0000 0.1408

Median

1,9776 0.00000 0.0000 0.4777

Half

2.6977 0.14123 0.2709 1.0865

75th percentile

3.7543 0.09422 0.1256 1,5096

Maximum

10.4061 4.24837 7.4410 8.1230

 GA%

GC% GG% GT%

Minimum

0.00000 0.0000 0.00000 0.0000

25th percentile

0.08696 0.0000 0.00000 0.9372

Median

0.32616 0.0000 0.00000 2,2408

Half

0.63347 0.2104 0.14123 3.2117

75th percentile

0.83333 0.1914 0.09422 4.7718

Maximum

5.77889 9.8795 4.24837 12.9479

TA%

 TC% TG% TT%

Minimum

28.63 0.00000 0.0000 0.000

25th percentile

33.48 0.08696 0.9695 4,234

Median

35.22 0.32616 1,9776 7,843

Half

35.29 0.63347 2.6977 7,184

75th percentile

37.14 0.83333 3.7543 10,110

Maximum

50.00 5.77889 10,406 17,290

Considering the results of the predicted S / MAR elements and the non-S / MAR sequences in the summary tables, notable differences can be seen in the AT and TA dinucleotide contents between these two groups of sequences. AT and TA respectively represent at least 18.5% and 28.6% of the dinucleotide content of the predicted S / MAR sequences, while the minimum percentages of the same dinucleotides in non-S / MAR sequences are respectively 0, 3% and 0%. Similarly, the maximum content of CC and GG in S / MAR sequences is 4.2%, while in non-S / MAR sequences the percentages

5 of these two dinucleotides can be up to 20.8%.

The correlation between the percentages of the AT and TA dinucleotides and the maximum DNA curvature calculated by the SMAR Scan @ program is represented in Fig. 17 for the predicted S / MAR sequences and in Fig. 18 for the sequences that were not are from S / MAR. The different scatterplots in these figures show that the TA percentage is well correlated with the DNA curvature predicted by the SMAR Scan® program.

10 Table 5: Summary of the percentages of dinucleotides in 1757 non-S / MAR sequences

AA%

AC% AG% AT%

Minimum

0.000 1,735 1,512 0.3257

25th percentile

7,096 4,586 6,466 5,1033

Median

9,106 5,016 7,279 6.8695

Half

8,976 5,054 7,184 7.0108

75th percentile

10,939 5,494 7,969 8.7913

Maximum

17,922 13,816 12,232 23.1788

 AC%

DC% CG% CT%

Minimum

3,571 0.8278 0.0000 1,512

25th percentile

6,765 4,1077 0.4727 6,466

Median

7,410 5.5556 0.8439 7,279

Half

7,411 5.9088 1,2707 7,184

75th percentile

8,010 7.2460 1.5760 7,969

Maximum

15,714 20.8415 12.6074 12,232

 GA%

GC% GG% GT%

Minimum

1,319 0.4967 0.8278 1,735

25th percentile

5,495 3.2615 4,1077 4,586

Median

6,032 4,4092 5.5556 5,016

Half

6,065 4.7468 5.9088 5,054

75th percentile

6,602 5.8824 7.2460 5,494

Maximum

10,423 16,0000 20.8415 13,816

TA%

TC% TG% TT%

Minimum

0.000 1,319 3,571 0.000

25th percentile

3,876 5,495 6,765 7,096

Median

5,625 6,032 7,410 9,106

Half

5,774 6,065 7,411 8,976

75th percentile

7,464 6,602 8,010 10,939

Maximum

24,338 10,423 15,714 17,922

Four of the new super MARs were chosen at random, the content of AT and TA dinucleotides was analyzed and compared with previously known chicken lysMARs, considering windows of 100 base pairs (Table 6).

Surprisingly, the inventors have found that all super MARs have AT dinucleotide frequencies greater than 12%, and TA dinucleotide frequencies greater than 10% of total dinucleotides.

15 analyzed in a 100 base pair DNA window. The most efficient MARs have values of around 34% of the two pairs of dinucleotides.

Table 6. Summary of frequencies in% of AT and TA dinucleotides of experimentally verified MAR

cLysMAR (mean of the CUE)

AT%: 12.03 TA%: 10.29 SEQ ID No

P1 68

AT%: 33.78 TA%: 33.93 SEQ ID No

P1 6

AT%: 34.67 TA%: 34.38 SEQ ID No

P1 42

AT%: 35.65 TA%: 35.52 SEQ ID No

Average value for all human "super" MARs

AT%: 34.32 TA%: 35.29

Average value for all those that are not "super" human MAR

AT%: 7.01 TA%: 5.77

6.4 Analysis of orthologous intergenic regions of the human and mouse genomes.

In order to obtain an understanding of the evolution of S / MAR, orthologous intergenic regions of human and mouse genomes have been analyzed with the SMAR Scan® program. The data set used is composed of 87 pairs of complete orthologous intergenic regions of the human and mouse genomes (Shabalina SA, Ogurtsov AY, Kondrashov VA, Kondrashov AS, "Selective constraint in intergenic regions of human and mouse genomes", Trends Genet., 17 (7): 3736, 2001) (mean length -12,000 bp) located on chromosomes human 12 and mouse 12. Tuning of these sequences was confirmed by base pair sequence alignment and consideration of flanking gene annotations (experimental or predicted).

The analysis of the 87 human and mouse orthologous intergenic sequences has been carried out with the SMAR Scan® program using their default settings. Analysis of human sequences yielded a total of 12 predicted S / MARs (representing a total length of 4,750 bp), located in 5 different intergenic sequences.

Among the three human intergenic sequences for which they are predicted to contain a "super" S / MAR using the restrictive settings of the SMARScan @ program, a corresponding orthologous mouse intergenic sequence containing a S / MAR is also predicted (human EMBL ID: Z96050, positions 28,010 to 76,951 orthologous to the mouse EMBL ID: AC015932, positions 59,884 to 89,963). When performing a local alignment of these two orthologous intergenic sequences, the best local alignment of these two large regions corresponds to the regions that the SMAR Scan® program predicts to be the S / MAR element. Using the Ensembl Genome Browser program (hftp: //ensembl.org), a manual search was performed for the mouse orthologous sequences of the other two human intergenic sequences that were predicted to contain a "super" S / MAR. The mouse orthologous intergenic sequences of these two human sequences were retrieved using orthologous predictions by the Ensembl program (based on gene names), searching for orthologous mouse genes for the human gene pairs flanking these intergenic regions.

Because the SMAR Scan® program has been fine-tuned for human sequences and consequently provides few "super" MARs with mouse genomic sequences, its default exclusion values were relatively relaxed for the minimum size of contiguous matches that are considered S / SEA (using 200 bp instead of 300 bp). Analysis by the SMAR Scan @ program of these mouse sequences predicted several S / MARs that have high values for the different calculated structural features. This finding suggests that elements of human MAR are conserved across species.

Example 7: Dissection of the 5'-MAR from the chicken lysozyme gene

The 3000 base pair 5'-MAR was dissected into smaller fragments that were monitored to effect transgene expression in Chinese hamster ovary (CHO) cells. To do so, seven ~ 400 bp fragments were generated by polymerase chain reaction (PCR). These PCR amplified fragments were contiguous and covered the entire MAR sequence when placed end-to-end. Four copies of each of these fragments were ligated in head-to-tail orientation, to obtain a length corresponding to approximately half of the natural MAR. The tetramers were inserted towards the 5 'end of the SV 40 promoter in pGEGFP Control, a modified version of the pGL3 Control vector (Promega). The plasmid pGEGFP Control was created by swapping the luciferase gene from pGL3 Control for the EGFP gene from pEGFP-N1 (Clontech). Plasmids containing the 5'-MAR fragment thus created were co-transfected with the resistance plasmid pSVneo in CHO-DG44 cells using LipofectAmine 2000 (Invitrogen) as a transfection reagent, as previously performed (Zahn-Zabal, M. , et al., "Development of stable cell lines for production or regulated expression using matrix attachment regions", J. Biotechnol., 2001. 87 (1): p. 29-42.). After selection of antibiotic resistant (G-418) cells, polyclonal cell populations were analyzed by FACS for EGFP fluorescence.

Transgene expression was expressed as the percentile of high-expression cells, defined as cells whose fluorescence levels are at least 4 orders of magnitude higher than the average fluorescence of cells transfected with the pGEGFP Control vector without MAR. Fig. 5 shows that multimerized fragments B, K and F increase the expression of transgenes, despite their shorter size compared to the original MAR sequence. In contrast, other fragments are either poorly active or completely inactive.

Example 8: Specificity of regions B, K and F in the MAR context

5'-MAR was serially deleted from the 5 'end (Fig. 6, top) or from the 3' end (Fig. 6, bottom), respectively. The effect of the truncated elements was monitored in a test similar to that described in the previous section. Figure 6 shows that the loss of ability to stimulate transgene expression in CHO cells was not uniformly distributed.

In this deletion study, the loss of MAR activity coincided with discrete transition regions that overlap with the 5'-MAR fragments B, K and F, respectively. In deletions at the 5 'end, activity was mostly lost when fragments K and F were removed. Deletions at the 3' end that removed elements F and B had the most pronounced effects. In contrast, flanking regions A, D, E and G had little or no ability to stimulate transgene expression by themselves (Fig. 5), and consequently did not contribute to MAR activity in deletion studies in the 5 'and 3' ends (Fig. 6).

Example 9: Structure of element F.

The 465 bp F fragment was further dissected into smaller 234, 243, 213 bp and 122, 125 and 121 bp sub-fragments, respectively. Fragments from the first group were octamerized (8 copies) in a head-to-tail orientation, while those from the second group were similarly hexadecamerized (16 copies), to maintain a constant length of the MAR sequence. These elements were cloned into the pGEGFP Control vector and their effects were analyzed in CHO cells as previously described. Interestingly, the FIII fragment retained most of the activity of the full-length F fragment, while the FII fragment containing the right side of the FIII fragment lost all ability to stimulate transgene expression (Fig. 7). This suggests that the active region comprised between 132 nucleotides and 221 nucleotides in the FIB fragment. Consequently, multiple copies of the FI and FIB fragments, spanning this region, exhibited similar activity. FIIA by itself has no activity. However, when added to FIB, resulting in FIII, it enhances the activity of the former. Therefore, FIIA appears to contain an ancillary sequence that has little activity by itself, but that reinforces the activity of the minimal domain located in FIB.

Analysis of the distribution of individual residues within the 5'-MAR of the lysozyme gene is shown in Fig. 8A, along with some additional residues that the inventors added to the analysis. Most of these remains were found to be scattered throughout the MAR element, and were not specifically associated with the active portions. For example, the sites of transcription factor binding and other residues that have been associated with MARs were not preferentially located in the active regions. It has also been proposed that active MAR sequences may consist of a combination of different residues. Various computer programs (MAR Finder, SMARTest, SIDD duplex stability) have been described to identify MARs as regions of DNA that associate with the DNA matrix. Such programs are usually based on algorithms that use a predefined series of sequence-specific models previously suggested to contain MAR activity, as illustrated by the MAR Finder program, currently known as the MAR Wiz program. The results obtained with these programs do not correlate well with the transcriptionally active portions of cLysMAR. For example, the activity peaks obtained with the MAR Finder program did not clearly coincide with the active MAR sub-portions, such as fragment B is quite active in vivo, but gives a negative score with the MAR Finder program (Fig. 8B, compare top and center panels). Curved DNA structures, as predicted by this program, did not correlate well with activity (Fig. 8B, compare top and bottom panels). Similar results were obtained with the other available programs (data not shown).

Therefore, it is unlikely that the residues identified by the available MAR prediction methods are the main determinants of the ability of cLysMARs to increase gene expression. Therefore, a number of other computer tools were analyzed. Surprisingly, it was found that the predicted nucleosome binding sequences and sequences that do not promote nucleosome binding were arranged in clusters interleaved repetitively in MARs, with sites that promote nucleosome binding overlapping with B, K regions. and F active. Nucleosome positioning sequences were proposed consisting of stretches of DNA that can easily envelop nucleosome histones, and which have not been previously associated with MAR sequences.

Sequences that promote nucleosome binding can be modeled using a set of DNA features that include moderately repeated sequences and other physico-chemical parameters that can allow the correct synchronization and orientation of DNA on the curved surface of histones. The identification of many of these DNA properties can be analyzed by computer, and up to 38 of these different properties have been used to predict potential nucleosome positions. Therefore, the inventors decided to determine if the specific components of nucleosome prediction programs could be correlated with the activity of MAR, with the aim of building a tool that allows the identification of new, and possibly more powerful, MAR from of genomic sequences.

To determine if some aspects of the primary DNA sequence could distinguish active B, K, and F regions from the surrounding MAR sequence, the inventors analyzed 5'-MARs with the MAR Scan® program. Of the 38 nucleosome disposition prediction tools, three were found that were correlated with the location of the active sub-domains of MAR (Fig. 9A). The location of the B, K and F regions in the MARs coincide with the maximum values of DNA curvature, the depth of the major groove and the width of the minor groove. A weaker correlation was also observed with the minimum values of the DNA fusion temperature, determined by its GC content. Fine-tuned mapping of the MAR fragment F indicated that the melting temperature valley and the top of the DNA curvature actually correspond to the FIB sub-fragment containing the MAR minimum domain (Fig. 9B). Therefore, the active portions of the MARs may correspond to the regions predicted by this program as curved DNA regions, and in the following text these regions will be referred to as CUE-B, CUE-K, and CUE-F. However, it is unknown whether these regions correspond to actual curved DNA and to base pair unwinding regions (BUR), since they do not correspond to curved DNA predicted by the MAR Wiz program (Fig. 9B).

Example 10: Traces of other regulatory elements in fragment F

The positioning characteristics of the nucleosome can be considered as one of the many chromatin-specific codes contained in genomic DNA. Although this particular code may contribute to the activity of the F region, it is unlikely to determine the activity of the MAR alone, since the 3 'part of the F region enhanced the activity of the MAR minimal domain contained in the FIB portion. Using the MatInspector program (Genomatix), the inventors searched for transcription factor binding sites with scores greater than 0.92 and found DNA binding sequences for the NMP4 and MEF2 proteins in the 3 'part of fragment F (Fig. 8B). To determine if some of these transcription factor binding sites might be located proximate to the active B and K regions, the complete sequence of 5'-MAR was analyzed to determine binding by NMP4, MEF2 and the proteins described to be bind to the single-chain or double-chain form of the BUR. Among these, SATB1 (AT-rich special binding protein 1) belongs to a class of DNA-binding transcription factor that can activate or suppress the expression of nearby genes. This study indicated that specific proteins such as SATB1, NMP4 (nuclear matrix protein 4) and MEF2 (myogenic enhancer factor 2) have a specific distribution and form a framework around the MAR minimal domains of cLysMAR (Fig. 10) . The presence of several of these NMP4 and SATB1 binding sites has been experimentally confirmed by EMSA analysis of purified recombinant proteins (data not shown).

Example 11: Construction of artificial MARs by combination of defined genetic elements

To further determine the relative roles of the various components of MAR, cLysMAR was deleted from all three CUE regions (Fig. 11, center part), resulting in the loss of part of its activity when compared to the sequence of Complete MAR similarly assembled from all its components as a control (Fig. 11, top). Consequently, one copy of each CUE alone, or one copy of the three head-to-tail assembled CUEs, had little activity in the absence of the flanking sequences. These results reinforce the conclusion that optimal transcriptional activity requires the combination of CUEs with flanking sequences. Interestingly, the entire MAR sequence generated from each of its components, but which also contains the Bglll-BamHl linker sequences (AGATCC) used to assemble each DNA fragment, exhibited high transcriptional activity (6-fold activation) in Comparison with the 4.8 times observed for the original MAR element in this series of tests (see Fig. 5).

The inventors investigated whether potentially curved DNA regions can also be active in a different environment than that found in their natural MAR context. Therefore, they decided to exchange the CUE-F, CUE-B and CUE-K elements, keeping the flanking sequences unchanged. The sequences flanking the CUE-F element were amplified by PCR and assembled to span the various CUEs, maintaining their original orientation and distance, or without a CUE. The ability of these genetically engineered ~ 1.8 kb MARs to increase transgene expression was then analyzed as above. All three CUEs were active in this context, and therefore action is not restricted to a given set of flanking sequences. Interestingly, the CUE-K element was even more active than CUE-F when inserted between the flanking sequences of CUE-F, and the first composite construct exhibited activity as high as that observed for full natural MAR (activation of 4 , 8 times). What distinguishes the CUE-K element from CUE-F and CUE-B is the presence of overlapping binding sites for the MEF-2 and SatB1 proteins, in addition to their CUE characteristic. Therefore, fusing CUE-B with the flanking domain of CUE-F results in a higher density of all three binding sites, which is probably the explanation for the increased activity. These results indicate that CUE assemblies with sequences containing binding sites for proteins such as NMP4, MEF-2, SatB1 and / or polyPpolyQ proteins constitute potent artificial MAR sequences.

Example 12: Expression Vectors

Three expression vectors according to the present invention are depicted in Figure 12.

Plasmid pPAG01 is a 5640 bp derivative of pUC19. It contains a 2960 bp chicken DNA fragment cloned into the BamH1 and XbaI restriction sites. This insert comes from the edge of the 5 'end of the chicken lysozyme locus and is high in A / T.

The control plasmid pGEGFP (also called pSV40EGFP) is a derivative of the pGL3-control vector (Promega) in which the luciferase gene sequence has been replaced by the form of the EGFP gene sequence from the pEGFP-N1 vector (Clontech) . The size of the plasmid pGEGFP is 4334 bp.

The control pUbCEGFP plasmid is a derivative of pGL3 with an ubiquitin promoter.

The plasmid pPAG01GFP (also called pMAR-SV40EGFP) is a derivative of pGEGFP with the 5'-LysMAR element cloned at the multiple cloning site (abbreviated MCS by the English expression Multiple Clonation Site) located just towards the 5 'end of the promoter of SV40. The size of the plasmid pPAG01EGF is 7285 bp.

Example 13: Effect of additional transfection of primary transfecting cells on expression of transgenes

One day before transfection, CHO-DG44 cells were grown in a 24-well plate, in growth medium at a density of 1.35 x 105 cells / well. 16 hours after the inoculum, the cells were transfected when they had reached 30-40% confluence, using Lipofect-AMINE 2000 (hereinafter LF2000), according to the manufacturer's instructions (Invitrogen). Twenty-seven microliters of serum-free medium (Opti-MEM; Invitrogen) containing 1.4 µl of LF2000 was mixed with 27 µl of Opti-MEM containing 830 ng of linear plasmid DNA. The antibiotic selection plasmid (pSVneo) amounted to one tenth of the reporter plasmid carrying the GFP transgene. The mixture was incubated at room temperature for 20 minutes, to allow the formation of the DNA-LF2000 complexes. The mixture was diluted with 300 µl of Opti-MEM and poured into previously emptied wells containing cells. After 3 hours incubation of the cells with the DNA mixture at 37 ° C in a CO2 incubator, a medium based on DMEM was added to each well. The cells were further incubated for 24 hours in a CO2 incubator at 37 ° C. The cells were then transfected a second time according to the method described above, except that the resistance plasmid carried another resistance gene (pSVpuro). Twenty-four hours after the second transfection, the 15 cells were passaged and expanded in a T-75 flask containing selection medium supplemented with 500 µg / ml G-418 and 5 µg / ml puromycin. After a two week selection period, the stably transfected cells were grown in 6-well plates. Alternatively, the cell population was transfected again using the same method, but with pTKhygro (Clontech) and pSVdhfr as resistance plasmids. GFP expression was analyzed with the Fluorescence Activated Cell Sorter (FACS) and with an apparatus

20 Fluoroscan.

Fig. 13 shows that the phenotype of the twice transfected cells (hereinafter referred to as secondary transfectants) was not only strongly colored, so that a special bulb and filter was not required to visualize the green color of the GFP protein , but also contained a majority of producer cells (FACS histogram in the lower left) compared to the parent population 25 (central histogram). This level of fluorescence corresponds to specific cell productivity of at least 10 pg per cell per day. Indeed, cells transfected only once (primary transfectants) that did not express the marker protein were almost entirely absent from the cell population after retransfection. Bars below 101 units of GFP fluorescence were 30% on the central histogram and less than 5% on the right histogram. This suggested that additional cells had

30 have been transfected and successfully expressed the GFP.

Surprisingly, the amount of fluorescence exhibited by the re-transfected cells suggested that the subpopulation of cells that had DNA incorporated twice expressed much more GFP than the expected double increase. Indeed the results shown in Table 2 indicate that the secondary transfectants exhibited, on average, an increase of more than twice the expected GFP if two sets of sequences, one in every 35 successive transfection, had been independently integrated and with similar efficiencies . Interestingly, this did not depend on the promoter sequence that activates the reporter gene, since both the viral and cellular promoter-containing vectors gave a similar increase in GFP (compare lanes 1 and 2). However, the effect was particularly marked for the vector containing the MAR compared to the plasmids without the MAR (lane 3), where the two consecutive transfections resulted in increases in the

Expression of 5.3 and 4.6 times, in two different experiments.

Table 7. Effect of re-transfecting primary transfectants at 24-hour intervals on GFP expression.

Plasmid type

Primary transfection Secondary transfection Increased PGFP fluorescence (times)

pUbCEGFP

4’992 14’334 2.8

pSV40EGFP

 4'324 12’237 2.8

pMAR-SV40EGFP

6’996 36’748 5.3

pUbCEGFP

6’452 15’794 2.5

pSV40EGFP

 4’433 11’735 2.6

pMAR-SV40EGFP

8’116 37’475 4.6

Two independent experiments are shown. The resistance plasmid pSVneo was co-transfected with various GFP expression vectors. One day after transfection, the cells were re-transfected with the same plasmids with the difference that the resistance plasmid was changed by pSVpuro. Cells bearing both resistance genes were selected in 500 µg / ml G-418 and 5 µg / ml puromycin and expression of the reporter gene marker was quantified by Fluoroscan. The number of times of magnification corresponds to the ratio between the fluorescence obtained from two consecutive transfections and the sum of the fluorescence obtained from the corresponding independent transfections. The number of times of increase that was considered significantly higher is shown in bold and corresponds to the fluorescence values that are

Consequently more than 2 times greater than the sum of those obtained from the independent transfections.

According to current knowledge, the increase in the level of GFP expression in multiple transfected cells was not expected, and this effect had not been previously observed.

Taken together, the data presented herein supports the idea that plasmid sequences that integrate primarily into the host genome would facilitate integration of other plasmids 5 by homologous recombination with the second incoming set of plasmid molecules. Plasmid recombination events occur within 1 hour after plasmid DNA has reached the nucleus and the frequency of homologous recombination between plasmid molecules co-injected into cultured mammalian cells has been shown to be extremely high, approaching unity [Folger, KR, K. Thomas, and MR Capecchi, "Non reciprocal exchanges of information between DNA duplexes coinjected into 10 mammalian cell nuclei". Mol. Cell Biol., 1985. 5 (1): p. 59-69], which explains the integration of multiple plasmid copies. However, homologous recombination between recently introduced DNA and its chromosomal counterpart normally occurs rarely, at a frequency of at most 1 in 103 cells receiving the DNA [Thomas, K.R., K.R. Folger, and M.R. Capecchi, "High frequency targeting of genes to specific sites in the mammalian genome", Cell, 1986. 44 (3): p. 419-28]. Therefore, the results could indicate that the element of

MAR works surprisingly to promote such recombination events. MARs would not only modify gene organization in vivo, and possibly also allow DNA replication along with viral DNA sequences, but could also act as DNA recombination signals.

Example 14: MARs mediate unexpectedly high expression levels in multiple transfected cells

20 If MAR-activated recombination events were to occur in multiple transfection methods, the inventors would expect that the synergy between the primary and secondary plasmid DNA would be affected by the presence of the MAR elements in one or both of the stages of transfection. The inventors examined this possibility by multiple transfections of the cells with pMAR alone or in combination with various expression plasmids, using the method previously described. Table 8 shows that transfecting cells twice with

The plasmid pMAR-SV40EGFP gave the highest expression of GFP and the highest degree of improvement under all conditions (4.3 fold). In contrast, transfecting the MAR-free vector twice gave little or no improvement, 2.8 fold, rather than the expected double increase. The inventors concluded that the presence of MAR elements at each transfection stage is necessary to achieve maximum protein synthesis.

Table 8

Primary transfection

Secondary transfection

Plasmid type

EGFP fluorescence  Plasmid type EGFP fluorescence Increase (times)

pMAR

0 pMAR pSV40EGFP pMAR-SV40EGFP 0 15’437 30’488 0 2.3-2.5 2.6-2.7

pMAR-SV40EGFP

 11’278 pMAR-SV40EGFP pMAR 47’027 12’319 4.3-5.3 1.0-1.1

pSV40EGFP

 6’114 pSV40EGFP pMAR 17’200 11’169 2.8 1.8-2.3

30 Interestingly, when cells were first transfected with pMAR alone, and then re-transfected with pSV40EGFP or pMAR-SV40EGFP, GFP levels were more than double compared to those resulting from a single transfection of subsequent plasmids (2 , 5 and 2.7 times respectively, instead of the expected 1 time). This indicates that previous transfection of MARs can increase the expression of the plasmid used in said second transfection method. Because MARs only act locally on the structure of the

For chromatin and gene expression, this implies that the two types of DNA may have been integrated into a similar chromosomal locus. In contrast, transfecting GFP expression vectors alone, followed by the MAR element in the second step, provided little or no improvement in GFP levels. This indicates that the order of transfection of the plasmids is important, and that the event of the first transfection must contain a MAR element that allows significantly higher levels of transgene expression.

If MAR elements favored homologous recombination of plasmids that remain in episomal forms from the first and second transfection methods, followed by their co-integration at a chromosomal locus, it would be expected that the order of plasmid transfection would not affect GFP levels. However, the above findings indicate that it is more favorable to transfect the MAR element in the first transfection event rather than in the second. This suggests the following molecular mechanism: during the first method

For transfection, the MAR elements can concatenate and integrate, at least in part, into the cell chromosome. This MAR DNA can in turn favor the subsequent integration of more plasmids, during the second transfection method, in the same chromosomal locus or in a nearby one.

Example 15: MARs as Agents Facilitating Long-Term DNA Transfer

If integrated MARs mediated a permissive chromosomal structure with persistent recombination, high levels of expression would be expected even if the second transfection was performed long after the first, at a time when most of the transiently introduced episomal DNA would have been removed . To address this possibility, the cells in Table 9, selected for their resistance to antibiotics for three weeks, were re-transfected once or twice and selected for the incorporation of additional DNA resistance markers. Cycles of tertiary, or tertiary and quaternary transfection, were performed with combinations of pMAR or pMAR-SV40EGFP, and analyzed for GFP expression as above.

Table 9. The MARs act as agents that facilitate the integration of DNA.

Tertiary transfection

Quaternary transfection

Plasmid type

EGFP fluorescence Times increase  Plasmid type EGFP fluorescence Times increase

pMAR

 18368 2.2 pMAR pMAR-SV40EGFP 43’186 140’000 2.4 7.6

pMAR-SV40EGFI

16544 2.0 pMAR- SV40EGFP pMAR 91’000 33’814 5.5 2.0

Secondary pMAR-SV40EGFP / pMAR-SV40EGFP transfectants were used in a third cycle of transfection at the end of the selection process. Tertiary transfection was performed with pMAR or pMAR-SV40EGFP and pTKhygro as the selection plasmid, to give tertiary transfectants. After 24 hours, the cells were transfected again with each of the plasmids and pSVdhfr, resulting in the quaternary transfectants that were selected in a growth medium containing 500 µg / ml of G-418, 5 µg / ml of puromycin, 300 μg / ml

15 of hygromycin B and 5 μM methotrexate. Secondary transfectants initially exhibited a GFP fluorescence of 8300. The number of times of increases corresponds to the ratio between the fluorescence obtained from two consecutive transfections and the sum of the fluorescence obtained from the corresponding independent transfections. The number of times of magnification that was considered significantly higher is shown in bold, and corresponds to the fluorescence values that are twice higher than the sum of those obtained from

20 independent transfections.

These results show that loading more copies of pMAR or pMAR-SV40EGFP resulted in similar double increases in total fluorescence of the cells. Loading even more MAR into the quaternary transfection increased this activity by another 2.4 fold. This is consistent with the inventors' hypothesis that recently introduced MAR sequences can be integrated into the chromosomal transgene locus by recombination.

They are homologous and therefore further increase the expression of transgenes.

When cells were transfected a third and fourth time with plasmid pMAR-SV40EGFP, GFP activity increased further, again at unexpected levels of the sum of fluorescence levels obtained from independent transfections. GFP expression reached levels that resulted in cells that visibly glowed green in daylight (Fig. 14). These results also indicate that the efficiency of

30 quaternary transfection was much greater than expected for the efficacy of the third DNA transfection, indicating that an appropriate time between transfections is crucial to obtain the optimal increase in gene expression, with one day being preferred to a period of three weeks.

The inventors believe that MAR elements favor secondary integration events by increasing the recombination frequency at their chromosomal integration site by relaxing the closed structure of chromatin, 35 since they mediate a local increase in histone acetylation (Yasui, D. , et al., "SATB1 targets chromatin remodeling to regulate genes over long distances", Nature, 2002. 419 (6907): p. 641-5.] Alternatively, or simultaneously, MARs potentially reposition nearby genes at sub locations -nuclear cells designed to be enriched in trans action factors, including proteins that can participate in recombination events, such as topoisomerases This can result in a locus at which sequences

MAR 40 can span the repeats of pSV40EGFP by efficiently protecting transgenes from chromatin mediated silencing effects.

Example 16: Use of MAR identified with the SMAR Scan® II program to increase the expression of a recombinant protein.

Four elements of MAR were randomly selected from the sequences obtained by sequence analysis

Complete the human genome with the SMAR Scan® program or the combined method. These MARs are called 1_6, 1_42, 1_68, (where the first number represents the chromosome from which the sequence originates and the second number is specific to the MAR predicted along this chromosome) and X_S29, a "super" MAR identified on the X chromosome. These predicted MARs were inserted into the pGEGFP Control vector towards the 5 'end of the SV40 promoter and enhancer that activates the expression of the green fluorescent protein and these plasmids.

50 were transfected into cultured CHO cells, as previously described (Zahn-Zabal, M., et al .; "Development of stable cell lines for production or regulated expression using matrix attachment regions", J.

Biotechnol., 2001. 87 (1): p. 29-42). Expression of the transgene was then analyzed in the total population of stably transfected cells using a fluorescence activated cell sorter (FACS). As can be seen in Fig. 19, all these recently identified MARs increased the expression of the transgene significantly above the expression activated by the chicken lysozyme Sea, the "super" MAR X_S29 being the most powerful of all the recently identified MARs .

Example 17: Effect on hematocrit of in vivo expression of mEPO by electrotransfer from the Network system with and without human MAR (1-68).

The therapeutic gene encodes EPO (erythropoietin), a hormone used to treat anemia. The EPO gene is placed under the control of a doxycycline inducible promoter, in a previously described gene exchange system called the Network system (Imhof, MO, Chatellard, P., and Mermod, N. (2000). " A regulatory network for efficient control of transgene expression ", J. Gene. Med. 2, 107-116.). The regulatory and EPO genes are then injected into the muscle of mice using the in vivo electroporation method called electrotransfer, so that the genes are transferred to the nuclei of the cells of the muscle fibers. When the antibiotic doxycycline is added to the drinking water of the mice, this compound is expected to induce the expression of EPO, which will lead to the elevation of the hematocrit level, due to the increase in the number of red blood cells mediated by the high level of circulating EPO. Therefore, if MAR improved EPO expression, higher hematocrit levels would be expected.

In vivo experiments were carried out on 5 week old C57BL6 female mice (Iffa Credo-Charles River, France). 30 µg of plasmid DNA was administered in normal saline by transcutaneous injections into the anterior muscle of the tibia. All injections were performed under anesthesia with ketaminol (75 mg / kg) and narcoxil (10 mg / kg). After intramuscular injection of DNA, an electric field was applied to the muscle. A voltage of 200 V / cm was applied in pulses of 8 milliseconds at 1 Hz (Bettan M, Darteil R, Caillaud JM, Soubrier F, Delaere P, Branelec D, Mahfoudi A, Duverger N, Scherman D. 2000 "High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. "Mol. Ther. 2: 204-10).

Sixteen mice were injected with the Network system that expresses EPO without MAR 1_68 and another 16 mice were injected with the Network system that incorporates MAR at the 5 'end of the promoter / enhancer sequences that activate the expression of the genes of the activator and EPO. In each group, half of the mice were administered doxycycline in the drinking water from the beginning of the experiment (day 0 - the day of the electrotransfer) and the other half, the doxycycline was put into the drinking water from on the 21st.

Blood samples were collected using heparinized capillaries by retro-orbital puncture at different times after injection of plasmids. The capillaries were centrifuged for 10 minutes at 5000 rpm at room temperature and the volume fraction of the red blood cells compared to the total blood volume was determined and expressed as a percentile, determining the hematocrit level.

As it can be deduced from Fig. 16 the group of mice injected by MAR-Network induced from the beginning of the experiment, presented a better induction of hematocrit compared to the mice injected with the original Network system without MAR. After 2 months, hematocrits in "groups containing MAR" were still higher (65%) than normal hematocrit levels (45-55%).

Most importantly, late induction (day 21) is possible only in the presence of MAR but in mice that were injected with the Network system without MAR. Thus, MAR probably protects transgenes from silencing and allows induction of their expression even after a prolonged period under non-inductive conditions.

Globally, the MAR element is capable of increasing the expression of the therapeutic gene, as detected by the increase in its physiological effect on the hematocrit.

SEQUENCE LIST

SEQUENCE LISTING

5 <110> Selexis S.A.

<120> High-efficiency gene transfer and expression in mammalian cells by a multiple transfection method of matrix-binding region sequences

10 <130> P2560EP04

<150> US 60 / 513,574

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15 <150> EP 08153753.2

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<170> PatentIn version 3.5

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<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (302)

<223> MAR of human chromosome 1, genomic contigs; 15088789 to 15089090

<400> 12 15

<210> 13

<213> Homo sapiens

<220>

<221> union_misc 25 <222> (1) .. (461)

<223> MAR of human chromosome 1, genomic contigs; 6791827 to 6792287

<400> 13

<210>

14 35 <211> 572

<212> DNA

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (572)

<223> MAR of human chromosome 1, genomic contigs; 163530 to 164101

<400> 14

<210>

15 10 <211> 357

36 <212> DNA

<213> Homo sapiens

<220> 15 <221> union_misc

<222> (1) .. (357)

<223> MAR of human chromosome 1, genomic contigs; 1842332 to 1842688

<400> 15 20

<210> 16

<213> Homo sapiens

<220>

<221>

union_misc 30 <222> (1) .. (399)

<223> MAR of human chromosome 1, genomic contigs; 2309560 to 2309958

<400> 16

<210> 17

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (394)

<223> MAR of human chromosome 1, genomic contigs; 2231759 to 2232152

<400> 17

<210> 18

<211> 387

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (387) 25 <223> MAR of human chromosome 1, genomic contigs; 7406524 to 7406910

<400> 18

<210> 19

<213> Homo sapiens <220>

<221> union_misc

<222> (1) .. (370)

<223> MAR of human chromosome 1, genomic contigs; 9399572 to 9399941

<400> 19

10 <210> 20

<211> 377

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (377)

<223> MAR of human chromosome 1, genomic contigs; 12417411 to 12417787

20 <400> 20

<210> 21 25 <211> 1524

<212> DNA

<213> Homo sapiens

<220> 30 <221> union_misc

<222> (1) .. (1524)

<223> MAR of human chromosome 1, genomic contigs; 1643307 to 1644830

<400> 21 35 <210> 22

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (664)

<223> MAR of human chromosome 1, genomic contigs; 1398763 to 1399426

<400> 22

15 <210> 23

<211> 1428 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (1428)

<223> MAR of human chromosome 2, genomic contigs; 17840365 to 17841792

<400> 23 <210> 24

<211> 4624 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (4624)

<223> MAR 1_6 of chromosome 1

<400> 24

<210> 25

<211> 3616 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (3616)

<223> MAR 1_68 on chromosome 1

<400> 25

<210> 26

<211> 4660 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (4660)

<223> MAR 1_42 on chromosome 1

<400> 26

<210> 27

<211> 3354 5 <212> DNA

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (3354)

<223> MAR X_S29 on chromosome x

<400> 27

<210> 28

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (677)

<223> MAR of chromosome 1 genomic contigs; 12803267..12803943

<400> 28

<210> 29

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (332)

<223> MAR of chromosome 1 genomic contigs; 13079684..13080015

<400> 29

<210> 30

<211> 479

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (479) 25 <223> MAR of chromosome 1 genomic contigs; 15682296..15682774

<400> 30

<210> 31

<211> 531

<212> DNA

<213> Homo sapiens <220>

<221> union_misc

<222> (1) .. (531)

<223> MAR of chromosome 1 genomic contigs; 15694611..15695141

<400> 31

<210> 32

<211> 378

<212> DNA

<213> Homo sapiens 15

<220>

<221> union_misc

<222> (1) .. (378)

<223> MAR of chromosome 1 genomic contigs; 886276..886653 20

<400> 32

25 <210> 33

<211> 595

<212> DNA

<213> Homo sapiens

30 <220>

<221> union_misc

<222> (1) .. (595)

<223> MAR of chromosome 1 genomic contigs; 3326732..3327326

35 <400> 33 <210> 34

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (738)

<223> MAR of chromosome 1 genomic contigs; 4485716..4486453

<400> 34

<210> 35

<211> 386

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (386) 25 <223> MAR of chromosome 1 genomic contigs; 5423067..5423452

<400> 35 <210> 36

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (584)

<223> MAR of chromosome 1 genomic contigs; 5805559..5806142

<400> 36

<210> 37

<211> 345

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (345) 25 <223> MAR of chromosome 1 genomic contigs; 10802644..10802988

<400> 37

<210> 38

<211> 474 <212> DNA

<213> Homo sapiens

<220> 5 <221> union_misc

<222> (1) .. (474)

<223> MAR of chromosome 1 genomic contigs; 13496468..13496941

<400> 38 10

<210> 39

<213> Homo sapiens

<220>

<221> union_misc 20 <222> (1) .. (483)

<223> MAR of chromosome 1 genomic contigs; 2509163..2509645

<400> 39

<210> 40

<211> 641

<212> DNA 30 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (641) 35 <223> MAR of chromosome 1 genomic contigs; 2776349..2776989

<400> 40 <210> 41

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (745)

<223> MAR of chromosome 1 genomic contigs; 2858703..2859447

<400> 41

<210> 42

<211> 307

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (307) 25 <223> MAR of chromosome 1 genomic contigs; 945522..945828

<400> 42 <210> 43

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (357)

<223> MAR of chromosome 1 genomic contigs; 3402743..3403099

<400> 43

<210> 44

<211> 323

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (323) 25 <223> MAR of chromosome 1 genomic contigs; 3485830..3486152

<400> 44

<210> 45

<213> Homo sapiens

<220>

<221> union_misc 40 <222> (1) .. (498)

<223> MAR of chromosome 1 genomic contigs; 3548336..3548833 <400> 45

5 <210> 46

<211> 400

<212> DNA

<213> Homo sapiens

10 <220>

<221> union_misc

<222> (1) .. (400)

<223> MAR of chromosome 1 genomic contigs; 4595109..4595508

15 <400> 46

<210> 47 20 <211> 403

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (403)

<223> MAR of chromosome 1 genomic contigs; 7205509..7205911

<400> 47 30

<210> 48 <211> 309

<212> DNA

<213> Homo sapiens

5 <220>

<221> union_misc

<222> (1) .. (309)

<223> MAR of chromosome 1 genomic contigs; 7507280..7507588

10 <400> 48

<210> 49 15 <211> 516

<212> DNA

<213> Homo sapiens

<220> 20 <221> union_misc

<222> (1) .. (516)

<223> MAR of chromosome 1 genomic contigs; 3581085..3581600

<400> 49 25

30 <210> 50

<211> 534

<212> DNA

<213> Homo sapiens

35 <220>

<221> union_misc

<222> (1) .. (534)

<223> MAR of chromosome 1 genomic contigs; 3084851..3085384

40 <400> 50 <210> 51

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (583)

<223> MAR of chromosome 1 genomic contigs; 160087..160669

<400> 51

<210> 52

<211> 314

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (314) 25 <223> MAR of chromosome 1 genomic contigs; 4350424..4350737

<400> 52

<210> 53

<211> 828

<212> DNA 5 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (828) 10 <223> MAR of chromosome 1 genomic contigs; 8443267..8444094

<400> 53

<210> 54

<211> 573

<212> DNA

<213> Homo sapiens 20

<220>

<221> union_misc

<222> (1) .. (573)

<223> MAR of chromosome 1 genomic contigs; 8703190..8703762 25

<400> 54

<210> 55

<211> 597

<212> DNA

<213> Homo sapiens 5

<220>

<221> union_misc

<222> (1) .. (597)

<223> MAR of chromosome 1 genomic contigs; 8819076..8819672 10

<400> 55

15 <210> 56

<211> 646

<212> DNA

<213> Homo sapiens

20 <220>

<221> union_misc

<222> (1) .. (646)

<223> MAR of chromosome 1 genomic contigs; 759619..760264

25 <400> 56

<210>

57 30 <211> 752

<212> DNA

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (752)

<223> MAR of chromosome 1 genomic contigs; 1226710..1227461

<400> 57

<210>

58 10 <211> 300

<212> DNA

<213> Homo sapiens

<220> 15 <221> union_misc

<222> (1) .. (300)

<223> MAR of chromosome 1 genomic contigs; 1119049..1119348

<400> 58 20

<210> 59

<211> 617 25 <212> DNA

<213> Homo sapiens

<220>

<221>

union_misc 30 <222> (1) .. (617)

<223> MAR of chromosome 1 genomic contigs; 3603613..3604229

<400> 59

<210> 60

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (674)

<223> MAR of chromosome 1 genomic contigs; 2592460..2593133

<400> 60

<210> 61 20 <211> 1694

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (1694)

<223> MAR of chromosome 1 genomic contigs; 2891680..2893373

<400> 61 30 5 <210> 62

<211> 587

<212> DNA

<213> Homo sapiens

10 <220>

<221> union_misc

<222> (1) .. (587)

<223> MAR of chromosome 1 genomic contigs; 3432560..3433146

15 <400> 62 <210> 63

<213> Homo sapiens

<220>

<221> misc_union 10 <222> (1) .. (313)

<223> MAR of chromosome 1 genomic contigs; 3805392..3805704

<400> 63

<210> 64 20 <211> 349

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (349)

<223> MAR of chromosome 1 genomic contigs; 4521378..4521726

<400> 64 30

<210> 65

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (500)

<223> MAR of chromosome 1 genomic contigs; 3240166..3240665

<400> 65

<210> 66

<211> 866

<212> DNA 15 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (866) 20 <223> MAR of chromosome 1 genomic contigs; 409429..410294

<400> 66

25 <210> 67

<211> 335

<212> DNA

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (335)

<223> MAR of chromosome 1 genomic contigs; 614754..615088

<400> 67

10 <210> 68

<211> 455

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (455)

<223> MAR of chromosome 1 genomic contigs; 1299520..1299974

20 <400> 68

<210> 69 25 <211> 404

<212> DNA

<213> Homo sapiens

<220> 30 <221> union_misc

<222> (1) .. (404)

<223> MAR of chromosome 1 genomic contigs; 1970778..1971181

<400> 69 35

<210> 70

<211> 605

<212> DNA 5 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (605) 10 <223> MAR of chromosome 1 genomic contigs; 3562918..3563522

<400> 70

<210> 71

<213> Homo sapiens

<220>

<221> union_misc 25 <222> (1) .. (317)

<223> MAR of chromosome 1 genomic contigs; 189743..190059

<400> 71

<210> 72

<211> 522

<212> DNA 35 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (522) 40 <223> MAR of chromosome 1 genomic contigs; 229111..229632

<400> 72 <212> DNA

<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (1110)

<223> MAR of chromosome 1 genomic contigs; 1138030..1139139

<400> 73

<210> 74

<211> 521

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

70 <222> (1) .. (521)

<223> MAR of chromosome 1 genomic contigs; 2863407..2863927

<400> 74

10 <210> 75

<211> 560

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (560)

<223> MAR of chromosome 1 genomic contigs; 5712303..5712869

20 <400> 75

<210> 76 25 <211> 479

<212> DNA

<213> Homo sapiens

<220> 30 <221> union_misc

<222> (1) .. (479)

<223> MAR of chromosome 1 genomic contigs; 8578812..8579290

<400> 76 35 5 <210> 77

<211> 477

<212> DNA

<213> Homo sapiens

10 <220>

<221> union_misc

<222> (1) .. (477)

<223> MAR of chromosome 1 genomic contigs; 8579294..8579770

15 <400> 77

<210> 78 20 <211> 331

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (331)

<223> MAR of chromosome 1 genomic contigs; 8580024..8580354

<400> 78 30

<210> 79

<213> Homo sapiens 72 <220>

<221> union_misc

<222> (1) .. (410)

<223> MAR of chromosome 1 genomic contigs; 8580705..8581114

<400> 79

<210> 80

<211> 433

<212> DNA

<213> Homo sapiens 15

<220>

<221> union_misc

<222> (1) .. (433)

<223> MAR of chromosome 1 genomic contigs; 12979167..12979599 20

<400> 80

25 <210> 81

<211> 385

<212> DNA

<213> Homo sapiens

30 <220>

<221> union_misc

<222> (1) .. (385)

<223> MAR of chromosome 1 genomic contigs; 16336644..16337028

35 <400> 81 <210> 82

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (363)

<223> MAR of chromosome 1 genomic contigs; 20624448..20624810

<400> 82

<210> 83

<211> 310

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (310) 25 <223> MAR of chromosome 1 genomic contigs; 566025..566334

<400> 83

<210> 84

<211> 1236

<212> DNA

<213> Homo sapiens 35

<220>

<221> union_misc

<222> (1) .. (1236)

<223> MAR of chromosome 1 genomic contigs; 1171429..1172664

<400> 84

10 <210> 85

<211> 309

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (309)

<223> MAR of chromosome 1 genomic contigs; 1925173..1925481

20 <400> 85

<210> 86 25 <211> 312

<212> DNA

<213> Homo sapiens

<220> 5 <221> union_misc

<222> (1) .. (312)

<223> MAR of chromosome 1 genomic contigs; 4396756..4397067

<400> 86 10

<210> 87

<213> Homo sapiens

<220>

<221> union_misc 20 <222> (1) .. (398)

<223> MAR of chromosome 1 genomic contigs; 56057..56454

<400> 87

<210> 88

<211> 391

<212> DNA 30 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (391) 35 <223> MAR of chromosome 1 genomic contigs; 56984..57374

<400> 88

<210> 89 5 <211> 309

<212> DNA

<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (309)

<223> MAR of chromosome 1 genomic contigs; 469547..469855

<400> 89 15

<210> 90

<213> Homo sapiens

<220>

<221> union_misc 25 <222> (1) .. (441)

<223> MAR of chromosome 1 genomic contigs; 546190..546630

<400> 90

<210> 91

<211> 1367

<212> DNA 35 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (1367) 40 <223> MAR of chromosome 1 genomic contigs; 124643..126009

<400> 91 <210> 92

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (458)

<223> MAR of chromosome 1 genomic contigs; 58908..59365

<400> 92

<210> 93 20 <211> 330

<212> DNA

78 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (330)

<223> MAR of chromosome 1 genomic contigs; 306867..307196

<400> 93

<210> 94

<211> 353

<212> DNA 15 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (353) 20 <223> MAR of chromosome 1 genomic contigs; 636899..637251

<400> 94

<210> 95

<211> 345

<212> DNA

<213> Homo sapiens 30

<220>

<221> union_misc

<222> (1) .. (345)

<223> MAR of chromosome 1 genomic contigs; 1435510..1435854 35

<400> 95

<210> 96

<211> 521

<212> DNA

<213> Homo sapiens

5 <220>

<221> union_misc

<222> (1) .. (521)

<223> MAR of chromosome 1 genomic contigs; 39695..40215

10 <400> 96

<210> 97 15 <211> 484

<212> DNA

<213> Homo sapiens

<220> 20 <221> union_misc

<222> (1) .. (484)

<223> MAR of chromosome 1 genomic contigs; 1286007..1286490

<400> 97 25

<210> 98

<213> Homo sapiens

<220>

<221> union_misc 35 <222> (1) .. (244)

<223> MAR of chromosome 1 genomic contigs; 73556..73879

<400> 98

<210> 99

<211> 463 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (463)

<223> MAR of chromosome 1 genomic contigs; 179038..179500

<400> 99

<210> 100

<211> 390

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (390) 25 <223> MAR of chromosome 1 genomic contigs; 55617..56006

<400> 100

<210> 101

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (582)

<223> MAR of chromosome 2 genomic contigs; 1157405..1157986

<400> 101

<210> 102

<213> Homo sapiens

<220>

<221> union_misc 15 <222> (1) .. (322)

<223> MAR of chromosome 2 genomic contigs; 1858638..1858959

<400> 102

<210> 103

<211> 914

<212> DNA 25 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (914) 30 <223> MAR of chromosome 2 genomic contigs; 5712196..5713109

<400> 103 <210> 104

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (370)

<223> MAR of chromosome 2 genomic contigs; 5713613..5713982

<400> 104

<210> 105

<211> 442

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (442) 25 <223> MAR of chromosome 2 genomic contigs; 7481647..7482088

<400> 105 <210> 106

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (338)

<223> MAR of chromosome 2 genomic contigs; 9594557..9594894

<400> 106

<210> 107

<211> 364

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (364) 25 <223> MAR of chromosome 2 genomic contigs; 10519720..10520083

<400> 107

<210> 108

<213> Homo sapiens <220>

<221> union_misc

<222> (1) .. (342)

<223> MAR of chromosome 2 genomic contigs; 11481943..11482284

<400> 108

10 <210> 109

<211> 415

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (415)

<223> MAR of chromosome 2 genomic contigs; 13499598..13500012

20 <400> 109

<210> 110 25 <211> 330

<212> DNA

<213> Homo sapiens

<220> 30 <221> union_misc

<222> (1) .. (330)

<223> MAR of chromosome 2 genomic contigs; 16370976..16371305

<400> 110 35

<210> 111

<211> 702 <212> DNA

<213> Homo sapiens

<220> 5 <221> union_misc

<222> (1) .. (702)

<223> MAR of chromosome 2 genomic contigs; 626641..627342

<400> 111 10

<210> 112

<213> Homo sapiens

<220>

<221> union_misc 20 <222> (1) .. (679)

<223> MAR of chromosome 2 genomic contigs; 3196047..3196725

<400> 112

<210> 113 30 <211> 728

<212> DNA 86

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (728)

<223> MAR of chromosome 2 genomic contigs; 3196778..3197505

<400> 113

<210> 114

<211> 413

<212> DNA

<213> Homo sapiens 15 <220>

<221> union_misc

<222> (1) .. (413)

<223> MAR of chromosome 2 genomic contigs; 2560638..2561050

<400> 114

<210> 115

<211> 361

<212> DNA 25 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (361) 30 <223> MAR of chromosome 2 genomic contigs; 4965309..4965669

<400> 115 87

<212> DNA

<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (325)

<223> MAR of chromosome 2 genomic contigs; 5258150..5258474

<400> 116 15

<210> 117

<211> 1508 20 <212> DNA

<213> Homo sapiens

<220>

<221>

union_misc 25 <222> (1) .. (1508)

<223> MAR of chromosome 2 genomic contigs; 6057499..6059006

<400> 117

<210> 118

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (415)

<223> MAR of chromosome 2 genomic contigs; 7996866..7997280

<400> 118

<210> 119

<211> 526

<212> DNA 5 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (526) 10 <223> MAR of chromosome 2 genomic contigs; 8300930..8301455

<400> 119

<210> 120

<211> 402

<212> DNA

<213> Homo sapiens 20

<220>

<221> union_misc

<222> (1) .. (402)

<223> MAR of chromosome 2 genomic contigs; 8576553..8576954 25

<400> 120

<210> 121

<211> 477

<212> DNA 35 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (477) 40 <223> MAR of chromosome 2 genomic contigs; 8785649..8786125

<400> 121 <210> 122

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (773)

<223> MAR of chromosome 2 genomic contigs; 10064737..10065509

<400> 122

<210> 123 20 <211> 1554

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (1554)

<223> MAR of chromosome 2 genomic contigs; 1039775..1041328

<400> 123 30 5 <210> 124

<211> 650

<212> DNA

<213> Homo sapiens

10 <220>

<221> union_misc

<222> (1) .. (650)

<223> MAR of chromosome 2 genomic contigs; 3944813..3945462

15 <400> 124 <210> 125

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (441)

<223> MAR of chromosome 2 genomic contigs; 5314265..5314705

<400> 125

<210> 126

<211> 1169

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (1169) 25 <223> MAR of chromosome 2 genomic contigs; 5953971..5955139

<400> 126 <210> 127

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (653)

<223> MAR of chromosome 2 genomic contigs; 6427669..6428321

<400> 127

<210> 128

<211> 414

<212> DNA

<213> Homo sapiens

5 <220>

<221> union_misc

<222> (1) .. (414)

<223> MAR of chromosome 2 genomic contigs; 10890453..10890866

10 <400> 128

<210> 129 15 <211> 496

<212> DNA

<213> Homo sapiens

<220> 20 <221> union_misc

<222> (1) .. (496)

<223> MAR of chromosome 2 genomic contigs; 13952568..13953063

<400> 129 25

30 <210> 130

<211> 317

<212> DNA

<213> Homo sapiens

35 <220>

<221> union_misc

<222> (1) .. (317)

<223> MAR of chromosome 2 genomic contigs; 16942865..16943181

40 <400> 130 <210> 131

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (464)

<223> MAR of chromosome 2 genomic contigs; 17217049..17217512

<400> 131

<210> 132

<211> 430

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (430) 25 <223> MAR of chromosome 2 genomic contigs; 19647266..19647695

<400> 132

<210> 133

<211> 2131

<212> DNA

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (2131)

<223> MAR of chromosome 2 genomic contigs; 20481223..20483353

<400> 133

<210> 134

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (842)

<223> MAR of chromosome 2 genomic contigs; 20483478..20484319

<400> 134

<210> 135 20 <211> 645

<212> DNA

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (645)

<223> MAR of chromosome 2 genomic contigs; 20897566..20898210

<400> 135

<210> 136

<211> 722

<212> DNA 15 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (722) 20 <223> MAR of chromosome 2 genomic contigs; 21664541..21665262

<400> 136

<210> 137

<213> Homo sapiens 99 <220>

<221> union_misc

<222> (1) .. (305)

<223> MAR of chromosome 2 genomic contigs; 22834991..22835295

<400> 137

<210> 138

<211> 352

<212> DNA

<213> Homo sapiens 15

<220>

<221> union_misc

<222> (1) .. (352)

<223> MAR of chromosome 2 genomic contigs; 25277762..25278113 20

<400> 138

25 <210> 139

<211> 342

<212> DNA

<213> Homo sapiens

30 <220>

<221> union_misc

<222> (1) .. (342)

<223> MAR of chromosome 2 genomic contigs; 25378452..25378793

35 <400> 139

<210> 140 40 <211> 663

<212> DNA 100

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (663)

<223> MAR of chromosome 2 genomic contigs; 30209437..30210099

<400> 140

<210> 141

<211> 1200

<212> DNA 15 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (1200) 20 <223> MAR of chromosome 2 genomic contigs; 31725089..31726288

<400> 141 <210> 142

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (325)

<223> MAR of chromosome 2 genomic contigs; 32147252..32147576

<400> 142

<210> 143

<211> 507

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (507) 25 <223> MAR of chromosome 2 genomic contigs; 32312662..32313168

<400> 143 <210> 144

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (339)

<223> MAR of chromosome 2 genomic contigs; 33651118..33651456

<400> 144

<210> 145

<211> 461

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (461) 25 <223> MAR of chromosome 2 genomic contigs; 45073053..45073513

<400> 145

<210> 146

<211> 1162 <212> DNA

<213> Homo sapiens

<220> 5 <221> union_misc

<222> (1) .. (1162)

<223> MAR of chromosome 2 genomic contigs; 45487691..45488852

<400> 146 10

<210> 147

<213> Homo sapiens

<220>

<221>

union_misc 20 <222> (1) .. (562)

<223> MAR of chromosome 2 genomic contigs; 45516233..45516794

<400> 147

<210> 148

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (801)

25 <223> MAR of chromosome 2 genomic contigs; 45727251..45728051

<400> 148

<210> 149

<211> 346

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (346) 25 <223> MAR of chromosome 2 genomic contigs; 50937238..50937583

<400> 149 <210> 150

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (462)

<223> MAR of chromosome 2 genomic contigs; 55672627..55673088

<400> 150

<210> 151

<211> 401

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (401) 25 <223> MAR of chromosome 2 genomic contigs; 56081352..56081752

<400> 151

30 35

<210> 152 <211> 765 <212> DNA <213> Homo sapiens <220>

106

<221> union_misc

<222> (1) .. (765)

<223> MAR of chromosome 2 genomic contigs; 56404208..56404972

<400> 152

<210> 153 10 <211> 443

<212> DNA

<213> Homo sapiens

<220> 15 <221> union_misc

<222> (1) .. (443)

<223> MAR of chromosome 2 genomic contigs; 61953416..61953858

<400> 153 20

<210> 154

<213> Homo sapiens

<220>

<221>

union_misc 30 <222> (1) .. (372)

<223> MAR of chromosome 2 genomic contigs; 62076211..62076582

<400> 154

<210> 155

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (484)

<223> MAR of chromosome 2 genomic contigs; 62158581..62159064

<400> 155

<210> 156

<211> 644

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (644) 25 <223> MAR of chromosome 2 genomic contigs; 68145036..68145679

<400> 156 <210> 157

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (530)

<223> MAR of chromosome 2 genomic contigs; 71257289..71257818

<400> 157

<210> 158

<211> 337

<212> DNA 20

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (337) 25 <223> MAR of chromosome 2 genomic contigs; 73413615..73413951

<400> 158 <210> 159

<211> 1340 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (1340)

<223> MAR of chromosome 2 genomic contigs; 77011049..77012388

<400> 159

<210> 160

<211> 937

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (937)

25 <223> MAR of chromosome 2 genomic contigs; 78226855..78227791 <400> 160

<210> 161

<211> 1350

<212> DNA

<213> Homo sapiens 10

<220>

<221> union_misc

<222> (1) .. (1350)

<223> MAR of chromosome 2 genomic contigs; 79287748..79289097 15

<400> 161 <210> 162

<213> Homo sapiens

<220>

<221> difference_misc 10 <222> (1) .. (332)

<223> MAR of chromosome 2 genomic contigs; 81142998..81143329

<400> 162

<210> 163

<211> 327

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (327) 25 <223> MAR of chromosome 2 genomic contigs; 84019536..84019862

<400> 163 <210> 164

<211> 407 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (407)

<223> MAR of chromosome 2 genomic contigs; 1448030..1448436

<400> 164

<210> 165

<211> 1959

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (1959) 25 <223> MAR of chromosome 2 genomic contigs; 2117630..2119588

<400> 165 <210> 166

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (520)

<223> MAR of chromosome 2 genomic contigs; 2119984..2120503

<400> 166

<210> 167

<211> 954

<212> DNA 5 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (954) 10 <223> MAR of chromosome 2 genomic contigs; 2578285..2579238

<400> 167

<210> 168

<211> 452

<212> DNA

<213> Homo sapiens 20

<220>

<221> union_misc

<222> (1) .. (452)

<223> MAR of chromosome 2 genomic contigs; 3836217..3836668 25

<400> 168

<210> 169

<211> 417

<212> DNA

<213> Homo sapiens 5

<220>

<221> union_misc

<222> (1) .. (417)

<223> MAR of chromosome 2 genomic contigs; 3837666..3838082 10

<400> 169

15 <210> 170

<211> 1197

<212> DNA

<213> Homo sapiens

20 <220>

<221> union_misc

<222> (1) .. (1197)

<223> MAR of chromosome 2 genomic contigs; 6294846..6296042

25 <400> 170 <210> 171

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (362)

<223> MAR of chromosome 2 genomic contigs; 6506971..6507332

<400> 171

<210> 172

<211> 2578

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (2578) 25 <223> MAR of chromosome 2 genomic contigs; 6507395..6509972

<400> 172

<210> 173

<211> 598

<212> DNA

<213> Homo sapiens 5

<220>

<221> union_misc

<222> (1) .. (598)

<223> MAR of chromosome 2 genomic contigs; 7770400..7770997 10

<400> 173

15 <210> 174

<211> 1048

<212> DNA

<213> Homo sapiens

20 <220>

<221> union_misc

<222> (1) .. (1048)

<223> MAR of chromosome 2 genomic contigs; 8332422..8333469

25 <400> 174 <210> 175

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (375)

<223> MAR of chromosome 2 genomic contigs; 8909678..8910052

<400> 175

<210> 176

<211> 563

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (563) 25 <223> MAR of chromosome 2 genomic contigs; 10572503..10573065

<400> 176 <210> 177

<211> 595 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (595)

<223> MAR of chromosome 2 genomic contigs; 11609694..11610288

<400> 177

<210> 178

<211> 662

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (662) 25 <223> MAR of chromosome 2 genomic contigs; 12699804..12700465

<400> 178 <210> 179

<211> 649 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (649)

<223> MAR of chromosome 2 genomic contigs; 12821904..12822552

<400> 179

<210> 180

<211> 3191

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (3191) 25 <223> MAR of chromosome 2 genomic contigs; 15356889..15360079

<400> 180

<210> 181

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (314)

<223> MAR of chromosome 2 genomic contigs; 728676..728989

<400> 181

<210> 182

<211> 423

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (423) 25 <223> MAR of chromosome 2 genomic contigs; 737493..737915

<400> 182 <210> 183

<211> 724 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (724)

<223> MAR of chromosome 2 genomic contigs; 1069556..1070279

<400> 183

<210> 184

<211> 383

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (383) 25 <223> MAR of chromosome 2 genomic contigs; 2719918..2720300

<400> 184

<210> 185

<211> 309

<212> DNA 5 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (309) 10 <223> MAR of chromosome 2 genomic contigs; 4994249..4994557

<400> 185

<210> 186

<211> 740

<212> DNA

<213> Homo sapiens 20

<220>

<221> union_misc

<222> (1) .. (740)

<223> MAR of chromosome 2 genomic contigs; 5034916..5035655 25

<400> 186

30

<210> 187 <211> 847 <212> DNA <213> Homo sapiens

35

<220> <221> union_misc <222> (1) .. (847)

126

<223> MAR of chromosome 2 genomic contigs; 6074678..6075524

<400> 187

<210> 188

<211> 784

<212> DNA 10 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (784) 15 <223> MAR of chromosome 2 genomic contigs; 6108986..6109769

<400> 188

<210> 189

<211> 381

<212> DNA

<213> Homo sapiens 5

<220>

<221> union_misc

<222> (1) .. (381)

<223> MAR of chromosome 2 genomic contigs; 10389032..10389412 10

<400> 189

15 <210> 190

<211> 507

<212> DNA

<213> Homo sapiens

20 <220>

<221> difference_misc

<222> (1) .. (507)

<223> MAR of chromosome 2 genomic contigs; 11097807..11098313

25 <400> 190

<210> 191

<211> 329

<212> DNA

<213> Homo sapiens 35

<220>

<221> union_misc

<222> (1) .. (329)

<223> MAR of chromosome 2 genomic contigs; 11234628..11234956 40

<400> 191 <210> 192

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (584)

<223> MAR of chromosome 2 genomic contigs; 797844..798427

<400> 192

<210> 193

<211> 363

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (363) 25 <223> MAR of chromosome 2 genomic contigs; 1093824..1094186

<400> 193

<210> 194

<211> 545

<212> DNA

<213> Homo sapiens

<220> 5 <221> union_misc

<222> (1) .. (545)

<223> MAR of chromosome 2 genomic contigs; 3456187..3456731

<400> 194 10

<210> 195

<213> Homo sapiens

<220>

<221> union_misc 20 <222> (1) .. (356)

<223> MAR of chromosome 2 genomic contigs; 5001567..5001922

<400> 195

<210> 196 30 <211> 321

<212> DNA

<213> Homo sapiens

<220> 35 <221> union_misc

<222> (1) .. (321)

<223> MAR of chromosome 2 genomic contigs; 5457330..5457650

<400> 196 40 <210> 197

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (361)

<223> MAR of chromosome 2 genomic contigs; 8124469..8124829

<400> 197

<210> 198

<211> 418

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (418) 25 <223> MAR of chromosome 2 genomic contigs; 11151485..11151902

<400> 198

<210> 199

<211> 394

<212> DNA

<213> Homo sapiens 35

<220>

<221> union_misc

<222> (1) .. (394)

<223> MAR of chromosome 2 genomic contigs; 13591477..13591870

<400> 199

<210> 200

<211> 1194 10 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 15 <222> (1) .. (1194)

<223> MAR of chromosome 2 genomic contigs; 14996824..14998017

<400> 200

<210> 201 25 <211> 487

<212> DNA 132

<213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (487)

<223> MAR of chromosome 2 genomic contigs; 14998429..14998915

<400> 201

<210> 202

<211> 421

<212> DNA 15 <213> Homo sapiens

<220>

<221> union_misc

<222>

(1) .. (421) 20 <223> MAR of chromosome 2 genomic contigs; 16562490..16562910

<400> 202

<210> 203

<220>

<221> union_misc

<222>

(1) .. (479) 35 <223> MAR of chromosome 2 genomic contigs; 21592301..21592779

<400> 203 <210> 204

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (870)

<223> MAR of chromosome 2 genomic contigs; 22557584..22558453

<400> 204

<210> 205 20 <211> 1086

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (1086)

<223> MAR of chromosome 2 genomic contigs; 30591960..30593045

<400> 205 30 <210> 206

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (406)

<223> MAR of chromosome 2 genomic contigs; 36233909..36234314

<400> 206

<210> 207

<211> 797

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (797)

25 <223> MAR of chromosome 2 genomic contigs; 36271745..36272541 <400> 207

5 <210> 208

<211> 423

<212> DNA

<213> Homo sapiens

10 <220>

<221> union_misc

<222> (1) .. (423)

<223> MAR of chromosome 2 genomic contigs; 36498521..36498943

15 <400> 208

<210> 209 20 <211> 304

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (304)

<223> MAR of chromosome 2 genomic contigs; 37179891..37180194

<400> 209 30 <210> 210

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (693)

<223> MAR of chromosome 2 genomic contigs; 38440448..38441140

<400> 210

<210> 211 20 <211> 471

<212> DNA

<213> Homo sapiens

<220> 25 <221> union_misc

<222> (1) .. (471)

<223> MAR of chromosome 2 genomic contigs; 38887582..38888052

<400> 211 30 <210> 212

<211> 1221 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (1221)

<223> MAR of chromosome 2 genomic contigs; 43885944..43887164

<400> 212

<210> 213 20 <211> 543

<212> DNA

<213> Homo sapiens <220>

<221> union_misc

<222> (1) .. (543)

<223> MAR of chromosome 2 genomic contigs; 45818200..45818742

<400> 213

10 <210> 214

<211> 463

<212> DNA

<213> Homo sapiens

15 <220>

<221> union_misc

<222> (1) .. (463)

<223> MAR of chromosome 2 genomic contigs; 47055478..47055940

20 <400> 214

<210> 215 25 <211> 2482

<212> DNA

<213> Homo sapiens

<220> 30 <221> union_misc

<222> (1) .. (2482)

<223> MAR of chromosome 2 genomic contigs; 47492696..47495177

<400> 215 35

<210> 216

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (539)

<223> MAR of chromosome 2 genomic contigs; 47561069..47561607

<400> 216 <210> 217

<211> 336 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (336)

<223> MAR of chromosome 2 genomic contigs; 52853648..52853983

<400> 217

<210> 218

<211> 406

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (406) 25 <223> MAR of chromosome 2 genomic contigs; 54866263..54866668

<400> 218

<210> 219

<211> 1452

<212> DNA

<213> Homo sapiens 35

<220>

<221> union_misc

<222> (1) .. (1452)

<223> MAR of chromosome 2 genomic contigs; 55113305..55114756

<400> 219

<212> DNA

<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (502)

<223> MAR of chromosome 2 genomic contigs; 56350637..56351138

<400> 220 15 <210> 221

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (794)

<223> MAR of chromosome 2 genomic contigs; 57051633..57052426

<400> 221

<210> 222

<211> 300

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (300) 25 <223> MAR of chromosome 2 genomic contigs; 57069272..57069571

<400> 222 <210> 223

<211> 370 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (370)

<223> MAR of chromosome 2 genomic contigs; 57235143..57235512

<400> 223

<210> 224

<211> 306

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (306) 25 <223> MAR of chromosome 2 genomic contigs; 57693125..57693430

<400> 224

<210> 225

<211> 500

<212> DNA

<213> Homo sapiens 35

<220>

<221> union_misc

<222> (1) .. (500)

<223> MAR of chromosome 2 genomic contigs; 59810331..59810830 40

<400> 225 145 <212> DNA

<213> Homo sapiens

<220> 10 <221> union_misc

<222> (1) .. (565)

<223> MAR of chromosome 2 genomic contigs; 59974589..59975153

<400> 226 15

<210> 227

<213> Homo sapiens

<220>

<221> union_misc 25 <222> (1) .. (427)

<223> MAR of chromosome 2 genomic contigs; 60605573..60605999

<400> 227 <210> 228

<211> 1199 5 <212> DNA

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (1199)

<223> MAR of chromosome 2 genomic contigs; 61229949..61231147

<400> 228

<210> 229

<211> 454

<212>

DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (454)

<223> MAR of chromosome 2 genomic contigs; 62181058..62181511

<400> 229

<210> 230

<211> 658

<212>

DNA 10 <213> Homo sapiens

147 5

<220>

<221> union_misc

<222> (1) .. (658) 15 <223> MAR of chromosome 2 genomic contigs; 62190919..62191576

<400> 230

<210> 231

<211> 1486

<212> DNA

<213> Homo sapiens 25

<220>

<221> union_misc

<222> (1) .. (1486)

<223> MAR of chromosome 2 genomic contigs; 62384127..62385612 30

<400> 231 <210> 232

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (333)

<223> MAR of chromosome 2 genomic contigs; 62538649..62538981

<400> 232

<210> 233 <211> 480

<212> DNA

<213> Homo sapiens

5 <220>

<221> union_misc

<222> (1) .. (480)

<223> MAR of chromosome 2 genomic contigs; 63240325..63240804

10 <400> 233

<210> 234 15 <211> 302

<212> DNA

<213> Homo sapiens

<220> 20 <221> union_misc

<222> (1) .. (302)

<223> MAR of chromosome 2 genomic contigs; 63935480..63935781

<400> 234 25

<210> 235

<213> Homo sapiens

<220>

<221>

union_misc 35 <222> (1) .. (407)

<223> MAR of chromosome 2 genomic contigs; 63935888..63936294

<400> 235

<210> 236

<213> Homo sapiens

<220>

<221>

union_misc 10 <222> (1) .. (302)

<223> MAR of chromosome 2 genomic contigs; 66958350..66958651

<400> 236

<210> 237

<211> 651

<212> DNA 20 <213> Homo sapiens

<220>

<221> union_misc

<222> (1) .. (651) 25 <223> MAR of chromosome 2 genomic contigs; 68307125..68307775

<400> 237

<210> 238

<211> 367

<212> DNA

<213> Homo sapiens 5

<220>

<221> union_misc

<222> (1) .. (367)

<223> MAR of chromosome 2 genomic contigs; 68308243..68308609 10

<400> 238

15 <210> 239

<211> 499

<212> DNA

<213> Homo sapiens

20 <220>

<221> union_misc

<222> (1) .. (499)

<223> MAR of chromosome 2 genomic contigs; 410241..410739

25 <400> 239

<210> 240 30 <211> 402

<212> DNA

<213> Homo sapiens

<220> 35 <221> union_misc

<222> (1) .. (402)

<223> MAR of chromosome 2 genomic contigs; 31531..31932

<400> 240 40 <210> 241

<213> Homo sapiens

<220>

<221> union_misc 10 <222> (1) .. (421)

<223> MAR of chromosome 2 genomic contigs; 32415..32835

<400> 241

Claims (16)

one.

An isolated and purified DNA sequence that has an activity that increases protein production, characterized in that said DNA sequence comprises a) at least one curved DNA element, where the curved DNA element contains at least 33% of the dinucleotide TA and / or at least 33% of the AT dinucleotide in a contiguous 100 base pair span, b) at least one binding site for a DNA binding protein, and because it is a MAR nucleotide sequence having the sequence SEQ ID No. 27, or one of its complementary sequences, or a sequence that has an identity of at least 90% with said sequence SEQ ID No. 27.

2.

The isolated and purified sequence according to claim 1, characterized in that said DNA-binding protein is a transcription factor preferably a transcription factor selected from the group comprising polyQpolyP domain proteins, or a transcription factor selected from the group comprising SATB1, NMP4, MEF2, S8, DLX1, FREAC7, BRN2, GATA 1/3, TATA, Bright, MSX, AP1, C / EBP, CREBP1, FOX, Freac7, HFH1, HNF3alfa, Nkx25, POU3F2, Pit1, TTF1 , XFD1, AR, C / EBPgamma, Cdc5, FOXD3, HFH3, HNF3 beta, MRF2, Oct1, POU6F1, SRF, V $ MTATA_B, XFD2, Bach2, CDP CR3, Cdx2, FOXJ2, HFL HP1, Myc, PBX, Pax TEF, VBP, XFD3, Brn2, COMP1, Evil, FOXP3, GATA4, HFN1, Lhx3, NKX3A, POU1F1, Pax6, TFIIA and Vmw65 or a combination of two or more of these transcription factors.

3.

In vitro use of an isolated and purified DNA sequence comprising a first nucleotide sequence isolated from the matrix-binding region (MAR) which is a MAR nucleotide sequence selected from the group comprising:

-

an isolated and purified DNA sequence according to claims 1 to 2,

-

SEQ ID No. 27, or one of its complementary sequences, to increase protein production activity in a eukaryotic host cell.

Four.

The use of the isolated and purified DNA sequence according to claim 3, characterized in that said isolated and purified DNA sequence further comprises a promoter operably linked to a gene of interest.

5.

The use of the isolated and purified DNA sequence according to claim 3 or 4, characterized in that said isolated and purified DNA sequence further comprises at least a second nucleotide sequence isolated from a matrix binding region (MAR) which is a MAR nucleotide sequence selected from the group comprising:

-

an isolated and purified DNA sequence according to claims 1 to 2,

-

SEQ ID No. 27, or one of its complementary sequences, to increase protein production activity in a eukaryotic host cell.

6.

The use of the isolated and purified DNA sequence according to claim 5, characterized in that said first and at least second MAR sequences are located at both 5 'and 3' ends of the sequence that contains the promoter and the gene of interest, or in a sequence other than that containing the promoter and the gene of interest.

7.

A method of in vitro transfection of a eukaryotic host cell, said method comprising a) introducing into said eukaryotic host cell at least one purified DNA sequence comprising at least one isolated and purified DNA sequence consisting of a MAR nucleotide sequence according to any of claims 1 or 2, b) subjecting in a defined time said transfected eukaryotic host cell to at least one additional step of transfection with at least one purified DNA sequence comprising at least one DNA sequence of interest, preferably a gene of interest encoding a protein operably linked to a promoter, and / or with at least one isolated and purified DNA sequence consisting of a MAR nucleotide sequence or other chromatin modifying elements, and c) selecting said transfected eukaryotic host cell, preferably select transfected eukaryotic host cells which e are highly protein-producing cells with a production rate of at least 10 pg per cell per day.

8.

The method according to claim 7, characterized in that the MAR nucleotide sequence is selected from the group comprising:

-

an isolated and purified DNA sequence according to claim 1 or 2,

-

the sequence SEQ ID No 27, or one of its complementary sequences.

9.

The method according to claim 7 or 8, characterized in that the defined time corresponds to intervals related to the cell division cycle.

10.

The method according to claim 9, characterized in that the defined time is the moment in which the host cell has just entered the second cycle of cell division.

eleven.

An in vitro method of transfection of a eukaryotic host cell, said method comprising cotransfecting into said eukaryotic host cell at least a first isolated and purified DNA sequence, which

5 comprises at least one DNA sequence of interest, and a second isolated and purified DNA sequence comprising at least one MAR nucleotide sequence selected from the group comprising:

-

an isolated and purified DNA sequence according to claim 1 or 2,

-

SEQ ID No. 27 or one of its complementary sequences.

12. A process for the production of a protein, where a) a transfected eukaryotic host cell of According to any one of claims 7 to 11, it is cultivated in a culture medium under conditions suitable for the expression of said protein and b) said protein is recovered. 13. A cellular transfection mix or kit comprising at least one isolated and purified DNA sequence according to claim 1 or 2. 14. A eukaryotic host cell transfected with at least one DNA sequence according to claim 1 or 2, and / or transfected with a method according to any one of claims 7 to 11.

fifteen.

A non-human transgenic organism, characterized in that at least one DNA sequence according to claim 1 or 2 is stably incorporated into its genome or at least some of its cells.

16.

The non-human transgenic organism according to claim 15, characterized in that some of its cells have been transfected according to the method according to any of claims 7 to 11.

 FIG. 2/1    FIG. 2/2    FIG. 2/3   FIG. 2/4