WO2004035780A1

WO2004035780A1 - Gene expression vector using echinus-origin insulators

Info

Publication number: WO2004035780A1
Application number: PCT/JP2003/013124
Authority: WO
Inventors: Satoshi Watanabe; Daisuke Honma; Hiroshi Yasue; Koji Akasaka; Kazuya Yoshida; Shingo Nagaya
Original assignee: National Institute Of Agrobiological Sciences; Bio-Oriented Technology Research Advancement Institution; Japan As Represented By The President Of The University Of Hiroshima
Priority date: 2002-10-16
Filing date: 2003-10-14
Publication date: 2004-04-29
Also published as: JP2004135532A; JP4300271B2

Abstract

It is attempted to develop a vector which shows stable expression while avoiding the inactivation of a transferred gene by using echinus-origin ARS gene insulators. Namely, vectors expressing a marker gene are constructed and transferred into cultured cells. Then functions of vectors having insulators ligated thereto are analyzed. As a result, it is revealed that, by ligating two insulators in a specific direction to an expression cassette, a gene can be stably expressed and the expression dose can be elevated 130-fold or more compared with the case with the ligation of no insulator. Thus, the efficaciousness of the thus constructed vector is clarified.

Description

TECHNICAL FIELD Gene expression vector using an insulator derived from Dini

The present invention relates to an expression vector for any DNA having an insulator. Background art

Eukaryotic genes consist of expression control regions such as enhancers and promoters and amino acid-encoding regions (structural genes). It is strictly determined that the promoter is located upstream of the structural gene. Enhansa, on the other hand, may be located upstream or downstream of a promoter or structural gene, and its position is not exactly determined. Some enhancers regulate gene expression from hundreds of kilobases away. It is known that Enhansa enhances the gene expression activity of the promoter by several times to several hundred times as its function.

In general, there are thousands of genes on one chromosome, and these genes are properly expressed with proper control. In other words, adjacent genes do not interfere with each other, and are not affected by unrelated genes. For this reason, it has been suggested that there may be a DNA sequence having a function (enhancer blocking) of cutting off the action of such an unrelated gene as an enhancer, and has been analyzed. In 1993, Dr. Felsenfeld and others at the NIH in the United States discovered DNA sequences responsible for such functions one after another from Drosophila and-ヮ, and named them "insulators." To date, insulators have been isolated from a variety of eukaryotes, including chickens, Drosophila, humans, and peni, and their functions have been analyzed. Among them, the insulator is not only involved in the enhancer blocking activity described above, but also plays the role of a shield that inhibits gene inactivation. It has become clear.

When genes on chromosomes do not need to be expressed according to cell type, etc., they are inactivated by chemical modifications such as methylation of DNA and deacetylation of histones, and structural changes such as heterochromatinization. Have been. Insulators protect genes from these inactivating effects. A specific example is a housekeeping gene. Housekeeping genes are essential genes for cell survival and are expressed independently of the inactivation of surrounding genes. It has become clear that insulators are present upstream, downstream, or at both ends of these genes and inhibit inactivation.

This shielding function has the potential to be very effective when performing gene recombination in cells including fertilized eggs. This is because the inactivation of the transgene may be suppressed. Genetic recombination in cells requires that genes be introduced into cells and that these vectors be integrated into the chromosome. However, even when the vector is integrated into the chromosome, it is affected by the surrounding DNA, and is inactivated by DNA methylation or deacetylation of histones, or the expression level is extremely small (position · Effect) has been announced.

So far, it has been suggested that connecting an insulator to a gene vector may avoid gene inactivation and stabilize transgene expression. So far, the insulator used for gene expression vectors is the HS4 insulator from chickens, and the HS4 insulator has a DNA length of 1.2 kb. Was difficult to use (Non-Patent Documents 1 to 4). In addition, there was no report that analyzed in detail the effect of the direction of ligation between an insulator and a vector on gene expression.

Prior art document information related to the invention of the application is shown below.

[Non-patent document 1] Walters MC, Fiering S, Bouhassira EE, Scalzo D, Goeke S, Magis W, Garrick D, Whitelaw E, Martin DI. The chicken beta—globin 5 'HS4 boundary element blocks enhancer-mediated suppression of silencing.Mol Cell Biol 1999 May; 19 (5): 3714- 26

(Non-Patent Document 2) Recillas-Targa F, Bell AC, Felsenfeld G. Positional enhancer-blocking activity of the chicken beta-globin insulator in transiently transfected cells.Proc Natl Acad Sci USA 1999 Dec 7; 96 (25): 14354 -9

[Non-Patent Document 3] Yannaki E, Tubb J, Aker M, Stamatoyannopoulos G, Emery DW.

Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors.Mol Ther 2002 May; 5 (5 Pt 1): 589-98

(Non-Patent Document 4) Rivella S, Callegari JA, May C, Tan CW, Sadelain M. The c HS4 insulator increases the probaoility of retroviral expression at rando m chromosomal integration sites.J Virol 2000 May; 74 (10): 4679- 87 Disclosure of the Invention

The present invention has been made in view of such circumstances, and an object of the present invention is to provide an expression vector for an arbitrary DNA having an insulator.

The present inventors have intensively studied to solve the above-mentioned problems. In 1999, Akasaka et al. Reported the use of a pen-derived ARS gene insulator to develop a vector that could stably express a transgene by avoiding inactivation. For this reason, we constructed a vector that expresses the gene and introduced it into cultured cells, and analyzed the function of the vector linked with an insulator. As a result, the gene can be stably expressed by connecting two insulators to the expression cassette in a specific direction, and the expression level is more than 130 times higher than when no insulator is connected. It became clear that This has clarified the effectiveness of the vector constructed by the present inventors.

That is, the present invention relates to an expression vector for an arbitrary DNA having an insulator, and provides the following [1] to [6]. [1] A vector having an insertion site for an arbitrary DNA, a promoter for controlling the expression of the arbitrary DNA, and an insulator, wherein the insulator is placed on the 3 ′ side of the insertion site for the arbitrary DNA. (4) A vector having forward and reverse directions on the 5 'side of a promoter that controls the expression of the arbitrary DNA.

[2] The vector according to [1], wherein the insulator is an insulator derived from a tree, a chicken or a Drosophila.

[3] The vector according to [1] or [2], wherein the insulator is a DNA according to any of the following (a) to (c):

(a) DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 3

(b) DNA consisting of the base sequence of any one of SEQ ID NOs: 1 to 3 wherein one or more bases are substituted, deleted, or added Z or inserted

(c) DNA that hybridizes to a DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 1 to 3 under stringent conditions

[4] The vector according to any one of [1] to [3], into which arbitrary DNA has been inserted. [5] A transformant carrying the vector according to any one of [1] to [4].

[6] A transformant in which the vector of [4] has been introduced into a chromosome.

The present invention provides a vector having an insertion site for an arbitrary DNA, a promoter for controlling the expression of the arbitrary DNA, an insulator, and an insulator, wherein the insulator is 3 ′ to the insertion site for the arbitrary DNA. A vector having forward and reverse directions is provided on each side of the promoter that controls the expression of the arbitrary DNA. By using such a vector, a transformant that expresses any DNA with high efficiency can be prepared.

In the present invention, the term “insulator” means a base sequence that functions as a shield for shielding the environment around the integrated genome when the vector is integrated into the genomic DNA of the cell. Examples of the insulator according to the present invention include, but are not limited to, ゥ -derived insulators, chicken-derived insulators, and Drosophila-derived insulators.

The above-mentioned insulators include a penis ARS gene-derived insulator (SEQ ID NO: 1), a chicken HS4 insulator (SEQ ID NO: 2), a peni-derived histone H2A insulator (SEQ ID NO: 3), and a Drosophila derived gypsy insulator

(Cai HN, Levine M. The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J 1997 Apr 1; 16 (7): 1732-41).

The peni ARS gene-derived insulator, the chicken HS4 insulator, the peni-derived histone H2A insulator, or the gypsy insulin-derived gypsy insulator can be prepared by methods known to those skilled in the art. For example, it can be isolated by performing Enhansaab opening screening on a DNA fragment cloned from a genomic genome, a chicken genome, or a Drosophila genome by PCR (Akasaka K, Nishimura A, Takata K, Mitsunaga K, Mibuka F, Ueda H, Hirose S, Tsutsui K, Snimada H. Upstream element of the sea urch in arylsulfatase gene serves as an insulator.Cell Mo 丄 Biol (Noisy-le-gra nd) 1999 Jul; 45 (5) : 555-65).

In addition, the insulator of the present invention includes DNA functionally equivalent to the DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 3. Here, “functionally equivalent” means that the target DNA has the same biological function (biological role) as the DNA consisting of the nucleotide sequence described in any of SEQ ID NOs: 1 to 3, and biochemistry. Has a functional function (biochemical activity). Examples of such a function include a function of inhibiting inactivation of any DNA introduced into a chromosome and an enhancer blocking activity. The expression “inhibits the inactivation of any DNA introduced into the chromosome” means that the expression of any DNA introduced into the chromosome is inhibited by methyl illness or the like. Also, the above Enhancer The term blocking activity refers to the activity of interrupting the action of the adjacent gene on the chromosome. Such functions and activities can be measured by enhancer blocking assay and Southern blotting by cleaving the genome of the transfected cells with a restriction enzyme sensitive to DNA methylation (Recillas- Targa F, Bell AC, Felsenfela G. Positional enhancer-blocking activity of the chicken beta-glob in insulator in transiently transfected cells. Proc Natl Aca d Sci USA 1999 Dec 7; 96 (25): 14354-9).

Other methods well known to those skilled in the art for preparing DNA functionally equivalent to certain DNAs include the replication technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9 58, Cold Spring Harbor Lab. Press, 1989). That is, those skilled in the art are aware of the penis ARS gene-derived insulator (SEQ ID NO: 1), the chicken-derived HS4 insulator (SEQ ID NO: 2), the mouse-derived histone H2A insulator (SEQ ID NO: 3), or It is a well-known technique to isolate DNA having a high homology with this by utilizing a part of the DNA.

In the present invention, a penis ARS gene-derived insulator, a chicken HS4 insulator, or a peni-derived histone H2A insulator under stringent conditions. It contains DNA that is functionally equivalent to the insulator or pentone-derived histone H2A insulator. The conditions of hybridization for isolating DNA functionally equivalent to the ARS gene-derived insulator, the chicken-derived HS4 insulator, or the chicken-derived histone H2A insulator are appropriately selected by those skilled in the art. can do. Hybridization conditions include, for example, low stringency conditions. Low stringency conditions include, for example, 42 ° C, 5X SSC, 0.1% SDS in washing after hybridization, and preferably 50 ° C, 5X SSC, 0.1%. This is the condition for SDS. More preferable conditions for the hybridization are high stringency conditions. High stringency conditions For example, the conditions are 65 ° C., 0.1 XSSC and 0.1% SDS. Under these conditions, it can be expected that DNA with higher homology can be obtained more efficiently as the temperature is increased. However, factors that affect the stringency of hybridization can be considered as multiple factors such as temperature and salt concentration, and those skilled in the art can realize the same stringency by appropriately selecting these factors. It is possible.

It was also synthesized based on the sequence information of the penis ARS gene-derived insulator (SEQ ID NO: 1), chicken-derived HS4 insulator (SEQ ID NO: 2), or peni-derived histone H2A insulator (SEQ ID NO: 3). Using primer-based gene amplification methods, such as the polymerase chain reaction (PCR) method, it is functionally equivalent to the penis ARS gene-derived insulator, the petri-derived HS4 insulator, or the peni-derived histone H2A insulator DNA can be isolated.

The nucleotide sequence of a DNA functionally equivalent to the peni ARS gene-derived insulator, the chicken HS4 insulator one, or the peni-derived histone H2A insulator isolated by these hybridization techniques or gene amplification techniques is SEQ ID NO: 1. To 3 in some cases. High homology usually means at least 80% identity, preferably 85% identity, more preferably 90% identity, and even more preferably 95% identity at the base level. Point.

The nucleotide sequence identity can be determined by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When a nucleotide sequence is analyzed by BLASTN based on BLAST, one parameter is, for example, score = 100 and wordlengt = 12. When using BLAST and Gapped BLAST programs, use the default parameters of each program. The specific methods of these analysis methods are public excuse (http: // www. Ncbi. Nlm. Nih. Gov.). In addition, one or more bases may be mutated (substitution, deletion, addition and Z or insertion) in the ARS gene-derived insulator sequence, the chicken-derived HS4 insulator sequence, or the peni-derived histone H2A insulator sequence. Thus, DNA functionally equivalent to the peni ARS gene-derived insulator, the chicken derived HS4 insulator, or the ゥ -derived histone H2A insulator is also included in the present invention. Such mutations can occur in nature. The number of bases to be mutated is usually within 30 bases, preferably within 15 bases, more preferably within 5 bases, and further preferably within 2 bases.

As a method well known to those skilled in the art for preparing DNA functionally equivalent to a certain DNA, a method of introducing a mutation into DNA is known. For example, those skilled in the art can use site-directed mutagenesis (Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods.Enzymol. 154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766), etc. By appropriately introducing a mutation into the sequence, a DNA functionally equivalent to the peni ARS gene-derived insulator, the chicken derived HS4 insulator, or the peni-derived histone H2A insulator can be prepared.

The insulator of the present invention may be in any form. That is, it does not matter whether it is genomic DNA or chemically synthesized DNA.

The vector of the present invention comprises the above-mentioned insulator on the 3 ′ side of the insertion site of any DNA in the DNA expression vector and on the 5 ′ side of the promoter controlling the expression of the arbitrary DNA, respectively, in the forward and reverse directions. It can be manufactured by purchasing. For example, as shown in Fig. 1, cDNA and poly A are added to any promoter such as pGK-LUC_p (A). A gene expression force set in which signals were linked was prepared in advance, and it was prepared using pBSK-SmBm (-) (Akasaka K, Nishimura A, TaKata K, Mitsunaga K, Mibuka F, Ueda H, Hirose S, Tsutsui K, Shimada H. Upstream element of the sea urchin arylsulfata se gene serves as an insulator.Cell Mol Biol (Noisy-le-grand) 1999 Jul; 5 (5): 555-65) And put it in the state of FR vector. Furthermore, after cutting the vector at the Kpnl site, Blunting is performed using T4 DNA polymerase, and the pBSK-SmBm (-) is excised with EcoRI-Sacl and the Blunted fragment is subcloned. Once the desired plasmid is obtained, the orientation of the insulator can be confirmed by cutting with Notl, and the appearance of a fragment of insulator + expression cassette + insulator. In addition, perform sequencing with an arbitrary primer to check the joint. Vectors can be produced by the above method. In the case of an existing vector, it is possible to change the vector to a connected vector by performing the same process. The present invention also provides a vector in which an arbitrary DNA has been inserted into the above vector. Examples of the arbitrary DNA of the present invention include those encoding any peptide, but the arbitrary DNA of the present invention is not limited thereto. Here, the term “peptide” refers to a compound in which amino acids are bound by peptide bonds. Therefore, “peptide” and “protein” which are peptides having a long chain length are also included in the “peptide” of the present invention. In the present invention, examples of the arbitrary peptide include an enzyme such as luciferase shown in the present application, a marker protein such as GFP, a cell functional protein such as a transcription factor, and a protein encoding a drug resistance gene. Force Any peptide of the present invention is not limited to these.

The present invention also provides a transformant in which a vector having any DNA of the present invention has been introduced into a chromosome.

The host into which the vector is introduced is not particularly limited, and for example, various eukaryotic cells and the like can be used. When using eukaryotic cells, e.g., animal cells, plant cells, etc. The vesicle can be used as a host. Animal cells include mammalian cells, for example, CH0, CO S., NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, for example, African oocyte (Valle, et. Al., Nature 291: 358-340, 19981) or insect cells such as Sf9, Sf21 and Tn5. As the CH0 cells, DHfr-CHO (Proc. Natl. Acad. Sci. USA, 1980, 77, 4216-4220.) And CHO K-l (Proc. Natl. Acad. Sci. USA, 1968, 60, 1275.) can be preferably used. Examples of plant cells include cells derived from Nicotiana tabacum.

As a method for introducing the vector of the present invention into the above host, in the case of introducing the vector into host cells such as cultured cells, for example, the calcium phosphate method (Chen, C. and 0kayama, H. Mol. Cell. Biol. , 1987, 7, 2745-2752.), DEAE dextran method (Lop ata, MA et. Al., Nucl. Acid. Res., 1984, 12, 5707-5717.), Sussman, DJ and Milman, G. Mol. Cell. Biol., 1985, 4, 1642-1643.), A method using catonic liposome D0TAP (Boehringer Mannheim), a lipofectin method (Derijard, B. Cell, 1994, 7, 1025-1037., Lamb, BT et. Al., Nature Genetics, 1993, 5, 22-30, Rabindran, SK et. Al., Science, 1993, 259, 230-234.) And the like. For introducing a vector into a plant cell, various methods known to those skilled in the art, such as a polyethylene glycol method, an electroporation method, a method via an agrobacterium, and a particle gun method, can be used.

The transformant in the present invention includes not only a transformed cell but also an individual having the transformed cell. For example, when using an animal as a host, mammals and insects can be mentioned. The mammal is not particularly limited, and for example, goats, puters, sheep, mice, mice, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Insects include, for example, silkworms, but are not limited thereto. Also, if you use plants, Tobacco can be used, but is not limited to this.

These animal and plant transformants can be produced by using known methods. For example, the target DNA is prepared as a fusion gene with a gene encoding a polypeptide uniquely produced in milk such as goat 3 casein. Then, a transgeneic goat can be produced by injecting a DNA fragment containing the fusion gene into a goat embryo and transplanting the embryo into a female goat (Ebert, KM et al. , Bio / Technology (1994) 12, 699-702).

In addition, transgenic silkworms can be produced, for example, by infecting chicks with a paculovirus into which DNA encoding the peptide of interest has been inserted (Susumu, M. et. Al., Nature, 1985, 315, 592—594.

In addition, for example, a DNA encoding the peptide of interest is introduced into a plant expression vector, for example, pMON530, and this vector is introduced into a pacteria such as Agrobacterium tumefaciens. Transgenic plants can be created by infecting this bacterium with tobacco, such as Nicotiana tapacam (Julian K. -C. Ma et. Al., Eur. J. Immunol., 1994, 24, 131-138.).

When the arbitrary DNA is a DNA encoding an arbitrary peptide, the peptide can be obtained, for example, from milk produced by the above-mentioned transjeckey or its progeny. It is also possible to recover peptides from cocoons and transgenic plants of transjeuc silkworms.

It is also possible to produce the above-mentioned transformed cells by culturing them. The culture can be performed according to a known method. For example, when culturing animal cells, generally, DMEM, MEM, RPMI1640, IMDM, or the like can be used as a culture solution. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. Usually, the pH during the culturing is preferably about 6 to 8. Culture is usually carried out at about 30 to 40 ° C for about 15 to 200 hours, and if necessary, the medium is replaced, aerated, and agitated. Add.

Any peptide can be purified as a substantially pure and homogeneous peptide. The separation and purification of the peptide may be carried out by using the separation and purification methods used in ordinary peptide purification, and is not limited at all. For example, chromatographic columns, filters, filtration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. If selected and combined, peptides can be separated and purified. Alternatively, purification can be performed by further combining a plurality of these columns. Examples of the chromatography include affinity chromatography in which an antibody to an arbitrary peptide is immobilized on a column, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography. Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Using these purification methods, any peptide can be highly purified.

The peptide can be arbitrarily modified or partially removed by reacting the peptide with a suitable peptide modifying enzyme before or after purification. Examples of peptide repair enzymes include, for example, trypsin, chymotrypsin, raised peptidase, protein kinase, dalcosidase and the like.

In addition, when any peptide is expressed in a host cell as a fusion peptide with glutathione S-transferase or as a recombinant peptide to which multiple histidines are added, the expressed recombinant peptide is daldal. After purifying the fusion peptide, if necessary, a region other than the target peptide in the fusion peptide may be purified using thrombin or a nickel column. It is also possible to cut and remove with factor Xa or the like.

The present invention also provides a transformant carrying the vector of the present invention. Such a transformant can be used, for example, for amplifying the vector of the present invention. The host into which the vector is introduced is not limited to the above eukaryotic cells, and prokaryotic cells can also be used. For example, prokaryotic cells include bacterial cells. Examples of the bacterial cells include Escherichia coli (E. coli), for example, J109, DH5a, HB101, XL1 Blue, BL21, and the like, and Bacillus subtilis. As a method for introducing the vector of the present invention into the above-mentioned host, in the case of introducing the vector into a host cell such as Escherichia coli, for example, a calcium chloride method and an electoporation method can be used. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the concept of the construction of a vector to which a peni-derived insulator was ligated. The gray arrow indicates the peni ARS gene insulator (580 pb). The direction of the arrow indicates that the insulator was ligated. PGK indicates mouse PGK-1 promoter LUC indicates luciferase gene, and SVp (A) indicates SV40 virus-derived polyA addition signal.

FIG. 2 is a diagram showing a gene expression vector using a penni-derived insulator. Parts constituting the vector are the same as those in FIG. The direction and number of insulators are different.

FIG. 3 is a diagram showing a gene expression vector using a penni-derived insulator. Parts constituting the vector are the same as those in FIG. The direction and number of insulators are different.

FIG. 4 is a diagram showing the results of measuring luciferase activity in cells into which the vector has been introduced. The luciferase activities of the 12 clones obtained for each vector are averaged and graphed. Luciferase activity of clones transfected with 0L vector It turns out to be very high. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Construction of a gene expression vector using a peni-derived insulator In order to clarify the function of the insulator, a bettater linked with a marker gene, a luciferase gene (LUC), was prepared. A method was used to clarify the function of the insulator using the expression level of the as an index.

As shown in FIG. 1, plasmid PBSK- SmBM with © two ARS gene derived insulator (580b _P) (-) to the Sail site, PGK-cut with Sail than PGK-luc plasmid LUC-p (A ) Subclosing the cassette (marker gene). For the obtained clone, confirm the direction with Hindlll and analyze the nucleotide sequence, and select the clone in which the PGK-LUC-p (A) cassette is connected in the opposite direction to the direction of the insulator. At the Kpnl site of the obtained clone, subclone an insulator slice cut out from pBSK-SmBm (-) with Sacl and EcoRI. By confirming the orientation with Notl and reading the nucleotide sequence of the obtained clone, a clone in which an insulator is ligated in the forward direction downstream of the PGK-LUC- _P (A) cassette is selected. The resulting clone is called the gene expression vector 0L (FIG. 2). In addition, seven types of FF, FR, RF, RR, IL, SL2, and SL3 (Fig. 3) were simultaneously constructed for the vectors prepared in the same manner, depending on the orientation and number of the insulators.

[Example 2] Introduction of vector into cells

As a mammalian cell, NIH3T3 cell, a mouse established fibroblast, was used as a model. NIH3T3 cells were cultured in DMEM medium (SIGMA) supplemented with 10% fetal calf serum. The NIH3T3 cells were transfected with a setter in which an insulator was linked to a marker gene, PGK-LUC-p (A), and a drug resistance gene, PGK-purop-p (A). Each vector In order to remove the plasmid region, the agarose gel was cut with restriction enzymes and subjected to agarose gel electrophoresis to cut out only the cassette site shown in Figs. The recovered cassette was mixed with 1/10 amount of PGK-purop-p (A) and introduced into cells using a gene transfer reagent ribofectamine plus (Invitrogen). 48 hours after the gene transfer, puromycin (SIGMA) was added to the medium at a concentration of 1 βg / ml, and the cells were cultured for 7 days to isolate recombinant cells. In addition, the recombinant cells were randomly isolated in 12 clones per vector.

When selection was performed with puromycin, no difference was observed between all vectors in the number of colonies of the obtained cells. In addition, 12 clones were isolated per vector for all vectors. Each clone was isolated on a 24-well microtiter plate, and when the cells grew, it was passed through two 12-well microtiter plates to make replicas. One of them was used for luciferase activity measurement, and the other was used for cell genomic DNA extraction and storage.

[Example 3] Lucifera Atsushi

For the clones obtained from each vector, genomic DNA was extracted and subjected to PCR to confirm the presence of the luciferase gene.

For the recombinant cells, the luciferase activity ^ 4 was measured using the luciferase Atsusei kit of PR0MEGA. At that time, the fluorescence intensity was measured using a diamond mouth CT-90000D as a measuring device. In addition, the amount of protein contained in each sample used for the measurement was measured using a protein assay kit manufactured by Amersham. For the data, the amount of fluorescence per protein was determined for each clone. In addition, based on these data, the average value of the amount of fluorescence of each vector was determined and simultaneously graphed (Fig. 4).

PGK-LUC-p (A), an unlinked insulator vector, showed an average of 441 activities, whereas FF, FR, and RF (Figure 3), each with one copy of an insulator, showed an activity of 441. The activity was 4672, 2585, 5687, and the activity was 5.9 to 12.9 times higher than that of PGK-LUC- _P (A) ^ 4. From these things, just linking one copy of the insulator, Despite the differences depending on the orientation and position of the insulator, it was clear that it had the ability to avoid gene inactivation.

When two copies of the insulator were ligated upstream and downstream of the cassette, IL, SL2, and SL3 (Fig. 3) exhibited activities of 3353, 1099, and 1377, respectively. This activity was 2.5 to 7.6 times the activity of PGK-LU C-p (A), and was found to be lower than when one copy of the insulator was ligated. However, at 0L (FIG. 1), the luciferase activity was 57800, which is 131 times that of PGK-LUC-p (A). From these facts, it became clear that when two copies of the insulator were ligated to the vector, the direction of expression caused a large difference in the gene expression level. In particular, it was found that connecting the insulator outwardly upstream and downstream of the force set showed extremely high transgene expression. These results were not seen at all in the other insulators reported so far, indicating that the penis ARS insulator is extremely effective as a shield to avoid gene inactivation. I have. In addition, it became clear that connecting an insulator such as 0L is effective as a means for expressing a gene with high efficiency when performing gene recombination.

So far, it has been suggested that the presence of two copies of an insulator in a vector may bind the insulators to form a loop, thereby inhibiting gene inactivation. Our results showed a difference in gene expression depending on the orientation of the insulator linked in two copies. It was suggested that the cause of such a difference was that it was difficult to form a loop depending on the direction of the insetter, and that there were combinations. Industrial potential

Genetic recombination using cultured cells or production of transgenic animals has been performed in many academic and basic research areas for drug development. Have been. However, in these transgenic experiments, it has been known that even when transgenic cells or transgenic animals are obtained, a phenomenon in which the introduced gene is not expressed may occur. Had one. As one of the causes of these phenomena, it has been revealed that the transgene is inactivated by receiving a position 'effect. However, until now there was no way to avoid this phenomenon. However, it has been clarified that by using the expression vector linked to the insulator developed in the present invention, inactivation of the gene can be avoided and the transgene can be expressed efficiently. This vector is very effective in the following research and development.

(1) Genetic recombination in cultured cell lines

When a foreign gene such as a virus is integrated into a chromosome as one of the intracellular immune functions, vertebrate cells can inactivate that gene by methylating DNA or deacetylating histones. Are known. For this reason, even if a gene expression vector is introduced for gene recombination, phenomena such as the fact that the transgene is not expressed or the amount of expression of the transgene is very small due to these effects may occur. It has been known. By using a vector to which an insulator is ligated, these can be avoided and a recombinant cell expressing the transgene efficiently can be obtained.

(2) Genetically modified animals

Genetically modified animals are extremely inefficient in their production because they are produced by directly injecting the gene expression vector DNA solution into the nuclei of fertilized eggs. Generally, the efficiency of obtaining an individual carrying the transgene is about 10% in mice and less than 1% in livestock. In addition, transgenic animals born at low efficiency are subject to inactivation of transgenes including position 'effects, so that only a small percentage of transgenes are expressed. By using a gene expression vector using an insulator, these phenomena can be avoided and the transgene can be expressed with high efficiency in transgenic animals.

Claims

The scope of the claims

1. A vector having an insertion site for an arbitrary DNA, a promoter for controlling the expression of the arbitrary DNA, and an insulator, wherein the insulator is located on the third side of the insertion site of the arbitrary DNA. A vector that has forward and reverse orientations on the 5, side of the promoter that controls DNA expression, respectively.

2. The vector according to claim 1, wherein the insulator is a pancreatic, chicken or Drosophila derived insulator.

3. The vector according to claim 1, wherein the insulator is a DNA according to any one of the following (a) to (c).

(a) DNA consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 3

(b) DNA consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted, added and / or inserted in the nucleotide sequence of any one of SEQ ID NOs: 1 to 3

4. The vector according to any one of claims 1 to 3, into which arbitrary DNA has been inserted.

5. A transformant carrying the vector according to any one of claims 1 to 4.

6. A transformant in which the vector according to claim 4 has been introduced into a chromosome.