JP4374454B2 - F9 embryonic tumor cells that can be used for protein production and use thereof - Google Patents

Description

  The present invention relates to F9 embryonic tumor cells that can be used at least for protein production and use thereof, and in particular, various proteins including laminin and recombinant proteins by modifying laminin α1 gene. The present invention relates to an F9 embryonic tumor cell that can be suitably used for the purpose of producing an extracellular matrix, secreted protein, and the like that incorporate sucrose in a large amount and at low cost, and a typical application technique thereof.

  The basement membrane is a structure having a thickness of about 100 nm containing laminin, type IV collagen, nidogen and perlecan as main components. In recent years, much evidence has been obtained that shows that the maintenance, proliferation, survival and differentiation of epithelial cells and parenchymal cells are controlled by the interaction between the basement membrane and cell surface receptor molecules.

  Laminin, one of the main constituent proteins of the basement membrane, is a heterotrimeric protein composed of three subunits of α chain, β chain, and γ chain. The existence of types (or four) and three types of isoforms is known. These combinations constitute laminin isoforms that exhibit various functions.

  Laminin-1 is α1β1γ1, laminin-2 is α2β1γ1, laminin-3 is α1β2γ1, laminin-4 is α2β2γ1, laminin-5 is α3β3γ2, laminin-6 is α3β1γ1, laminin-7 is α3β2γ1, laminin -8 is α4β1γ1, laminin-9 is α4β2γ1, laminin-10 is α5β1γ1, laminin-11 is α5β2γ1, laminin-12 is α2β1γ3, laminin-13 is α3β2γ3, laminin-14 is α4β2γ3, laminin-14 15 is each combination of α5β2γ3.

  As described above, laminin is composed of many laminin isoforms and is rich in diversity as compared with other basement membrane main constituent proteins. Thus, it has been suggested that each laminin isoform is an important molecule that defines basement membrane diversity. Indeed, these laminin isoforms show specific expression patterns depending on the stage of development in various tissues. In addition, knockout mice of each laminin subunit show a specific phenotype. The above indicates that each laminin isoform has a specific function in the control of cell proliferation, survival and differentiation.

  Some laminin isoforms are widely used in research and industrial fields. Therefore, a technique for stably and inexpensively adjusting a large amount of a basement membrane-like substrate containing a laminin isoform having physiological activity is required.

  Conventionally, as a technique for preparing the laminin isoform, a technique for directly purifying from a biological tissue or a cell culture solution is known (see Non-Patent Document 1 and Non-Patent Document 2).

  As a technique for preparing laminin-1, a technique using a mouse Engelbreth-Holm-Swarm (EHS) tumor is known. When the EHS tumor is used, a crude extract containing a basement membrane component at a high concentration (for example, a crude extract containing laminin-1 at about 3-10 mg / ml) can be easily obtained. This crude extract is widely used not only as a purification raw material for laminin-1 but also as a basement membrane-like substrate in various cell cultures and assays, and is a very important material in the fields of tissue engineering, developmental engineering, and regenerative medicine. (See Non-Patent Document 3).

Furthermore, in order to produce a desired laminin isoform, a technique for forcibly expressing various laminin subunits as recombinant proteins in cultured cells is also used. In this technique, human kidney-derived 293 cells or the like are used as cultured cells, and expression vectors having a CMV promoter, EF-1 promoter, or the like are used as expression vectors (see Non-Patent Documents 4 to 7).
Wewer UM et al, Methods Enzymol 1994; 245: 85-104 Kikkawa Y et al, J. Biol. Chem 1998,273,15854-9 Kleinman HK et al, Biochemistry, 1986, 25: 312-8 Kortesmaa J, J. Biol Chem, 2000,275: 14853-9 Doi M et al, J Biol Chem, 2002,277: 12741-8 Hayashi Y et al, Biochem Biophys Res Commun 2002,299: 498-504 Ido H et al, J. Biol. Chem, 2004, 279: 10946-54

  However, the above conventional technique has a problem that it is difficult to stably and inexpensively prepare a large amount of a basement membrane-like substrate containing a desired laminin isoform having physiological activity.

  First, with the technique of directly purifying from a living body as disclosed in Non-Patent Document 1, it is generally difficult to purify the components of the basement membrane including laminin. This is because the basement membrane thickness in the living body is maintained at a submicrometer level of about 100 nm in most tissues, and it is difficult to secure a sufficient amount of basement membrane-like substrate. .

  Further, in the technique using an EHS tumor as disclosed in Non-Patent Document 3, the EHS tumor is used as a purification raw material to purify laminin 1, but other laminin isoforms are almost expressed. Therefore, it cannot be said that it is suitable as a raw material for purifying other laminin isoforms.

  Furthermore, it is known that the technology for expressing and purifying laminin as a recombinant protein as disclosed in Non-Patent Documents 4 to 7 has poor efficiency in expression and purification of the recombinant protein. This is because laminin is a trimer composed of three subunits that undergo complex glycosylation. Moreover, since this purified laminin also contains physiologically inactive laminin, the extent to which active laminin is contained in the purified laminin is also a big problem in practice.

  The present invention has been made in view of the above-mentioned problems, and the object thereof is F9 embryo that can be suitably used for the purpose of efficiently mass-producing extracellular matrix and secreted protein incorporating various extracellular matrix molecules. It is to provide sex tumor cells and typical utilization techniques thereof.

  As a result of intensive studies in view of the above problems, the present inventors have incorporated an extracellular matrix molecule into the laminin α1 locus by inserting an exogenous gene so as to be expressed under the control of the laminin α1 gene promoter. The inventors have found that extracellular matrix and secreted protein can be efficiently produced, and have completed the present invention.

  That is, the F9 embryonic tumor cell according to the present invention is an F9 embryonic tumor cell that can be used at least for protein production in order to solve the above-described problems, and among the pair of laminin α1 loci, One gene locus is characterized in that, in place of the laminin α1 gene, an exogenous gene insertion sequence for inserting an exogenous gene so as to be expressed under the control of the laminin α1 gene promoter is provided. .

  In the F9 embryonic tumor cell, the laminin α1 gene is preferably disrupted at the other locus. Further, it is more preferable to insert a gene encoding a protein constituting a basement membrane or a secreted protein into the foreign gene insertion sequence. Here, examples of a gene encoding a protein constituting the basement membrane or a secreted protein include laminin α1, α2, α3, α4, and α5 genes.

  Further, the foreign gene insertion sequence preferably includes a marker gene, and as the marker gene, a gene encoding a fluorescent protein, a luciferase gene, or a secreted alkaline phosphatase gene is preferably used. Examples of genes encoding fluorescent proteins include EGFP gene, AcGFP gene, AmCyan gene, AsRed gene, DsRed gene, EBFP gene, ECFP gene, EYFP gene, HcRed gene, ZsGreen gene, and ZsYellow gene (Clontech). Can do.

  The present invention also includes a method for producing an extracellular matrix using the F9 embryonic tumor cells and a method for screening a cell differentiation inducing factor, differentiation inhibitory factor or anticancer agent using the F9 embryonic tumor cells.

  Furthermore, the present invention includes a method (production method) for producing the F9 embryonic tumor cell. Specifically, it is a method for producing an F9 embryonic tumor cell that can be used for at least protein production, in which one of the pair of laminin α1 loci is replaced with laminin α1 instead of the laminin gene. Examples thereof include a method including a step of incorporating a foreign gene insertion sequence for inserting a foreign gene so that the gene is expressed under the control of a gene promoter.

  In the production method, it is preferable that the method further comprises a step of inserting a gene encoding a protein constituting a basement membrane or a secreted protein into the foreign gene insertion sequence, and the other of the pair of laminin α1 loci. Preferably, the method comprises a step of disrupting the laminin α1 gene at the gene locus.

  As described above, in the present invention, in F9 embryonic tumor cells, an exogenous gene is inserted into or can be inserted into the laminin α1 gene locus under the control of the laminin α1 gene promoter. This makes it possible to express the desired gene under the control of the laminin α1 gene promoter. Therefore, expression of a desired gene can be induced by differentiation-inducing the F9 embryonic tumor cell with a differentiation-inducing agent or the like. As a result, there is an effect that it is possible to efficiently prepare a large amount of extracellular matrix and secreted protein incorporating various extracellular matrix molecules.

  An embodiment of the present invention will be specifically described as follows, but the present invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

(I) F9 embryonic tumor cell according to the present invention The F9 embryonic tumor cell according to the present invention (hereinafter sometimes simply referred to as "F9 cell") can be used at least for protein production. A foreign gene insertion sequence for inserting an exogenous gene so as to be expressed under the control of the laminin α1 gene promoter, instead of the laminin α1 gene, in one of the pair of laminin α1 gene loci Is provided.

[Laminin locus]
The laminin α1 locus is located on chromosome 17. The laminin α1 gene promoter can be activated by various stimuli. For example, the laminin α1 gene promoter can be activated by inducing differentiation of F9 embryonic tumor cells. Moreover, in the Example mentioned later, F9 embryonic tumor cells are induced to differentiate using retinoic acid and cAMP to activate the promoter of laminin α1 gene.

  In the present invention, in the laminin α1 gene locus, the foreign gene insertion sequence is incorporated into at least one gene locus, but the laminin α1 gene is preferably disrupted in the other gene locus. This completely eliminates the expression of the α1 subunit. Therefore, F9 − / − cells can be used as a laminin α1 knockout cell for functional analysis of the laminin α1 gene. In addition, for example, the production of laminin α1 subunit can be suppressed, and a laminin isoform having a desired configuration (for example, having an α chain other than laminin α1 subunit) can be efficiently produced. More specifically, for example, F9 embryonic tumor cells endogenously express laminin-1 (α1β1γ1). When an attempt is made to produce laminin-10 (α5β1γ1) by expressing laminin α5 subunit in the F9 embryonic tumor cell, endogenous β1 subunit and γ1 subunit are exogenously expressed α5 subunit. It forms a heterotrimeric protein with the α1 subunit expressed more endogenously. As a result, the production efficiency of desired laminin isoforms including laminin-10 could not be increased. However, as described above, a laminin isoform having an α chain other than the desired laminin α1 subunit can be efficiently produced by disrupting the gene of the laminin α1 subunit.

  Here, the above “disruption of laminin α1 gene” indicates that the production of laminin α1 subunit is inhibited. The specific method for destroying the laminin α1 gene is not particularly limited, and a conventionally known molecular biological method can be used. Specifically, as shown in the examples described later, a technique is adopted in which foreign sequences are inserted by homologous recombination and expression is suppressed by deleting a part of exons of the α1 subunit.

[Foreign gene insertion sequence]
As the foreign gene insertion sequence, a foreign gene is inserted into one of the pair of laminin α1 loci so that the gene is expressed under the control of the laminin α1 gene promoter instead of the laminin α1 gene. The gene sequence is not particularly limited as long as it is a gene sequence. Specific examples include lox sequences, Flp recombination target sequences (Invitrogen), and att related sequences (Invitrogen). At this time, CRE recombinase, Flp recombinase and gateway clonase can be used as the recombination enzyme, respectively, but any enzyme capable of causing recombination may be used, and it is not particularly limited.

  The gene inserted into the foreign gene insertion sequence is not particularly limited, and a wide variety of foreign genes can be inserted. For example, a gene encoding a protein constituting a basement membrane or a secreted protein can be mentioned. More specifically, for example, laminin α1, α2, α3, α4 or α5 gene can be mentioned.

  The foreign gene insertion sequence preferably further includes a marker gene. As such a marker gene, any marker gene can be used as long as it can be used as an index for determining the transcription state of the promoter of laminin α1 gene (whether transcription is activated or repressed). Can be used. Specifically, for example, an EGFP gene, an AcGFP gene, an AmCyan gene, an AsRed gene, a DsRed gene, an EBFP gene, an ECFP gene, an EYFP gene, an HcRed gene, a ZsGreen gene, a Zs Yellow gene, a luciferase gene, or a secreted alkaline phosphatase gene. Can be mentioned.

  By having the marker gene as described above, it can be used in the screening method described later.

[Other configurations]
The F9 embryonic tumor cell used in the present invention may be any one that can be used at least for protein production. Specifically, for example, as shown in Examples described later, F9 cells that can be obtained by a conventional method (for example, purchasing commercially available cells) can be suitably used.

  According to the above configuration, since the foreign gene insertion sequence for inserting the foreign gene is inserted so that the gene is expressed under the promoter control of the laminin α1 gene, the desired gene is expressed under the promoter control of the laminin α1 gene. It becomes possible to make it. In addition, F9 embryonic tumor cells according to the present invention in which a desired gene is inserted into the foreign gene insertion sequence can control the expression timing of the desired gene by inducing differentiation by retinoic acid and cAMP treatment or the like. Become. Furthermore, the F9 embryonic tumor cells before being induced to differentiate by treatment with retinoic acid and cAMP, etc. rapidly proliferate. For this reason, in order to obtain a large number of cells expressing the desired gene, F9 embryonic tumor cells in which the desired gene is inserted into the foreign gene insertion sequence are cultured in large quantities before differentiation induction, and then differentiation induction is performed. This makes it possible to obtain a large number of cells that express the desired gene. That is, if the F9 embryonic tumor cell of the present invention is used, a system for expressing a desired gene in a large amount can be easily scaled up.

(II) Method for Producing F9 Embryonic Tumor Cell According to the Present Invention The method (production method or production method) for producing F9 embryonic tumor cell according to the present invention is not particularly limited. Any method can be used as long as it can produce (produce or produce) the aforementioned F9 embryonic tumor cells. For example, specifically, in order to insert a foreign gene into one of the pair of laminin α1 loci so that the gene is expressed under the control of the laminin α1 gene promoter instead of the laminin gene. A step of incorporating a foreign gene insertion sequence (step of incorporating a foreign gene insertion sequence) may be included.

  In the foreign gene insertion sequence integration step, the above-described foreign gene insertion sequence may be incorporated into the laminin locus, but the specific method is not particularly limited, and a known gene recombination technique can be used in the present invention. That's fine. In the examples described later, the homologous recombination method is used. Furthermore, it is preferable to include a step of inserting a gene constituting a basement membrane or a secretory protein into the foreign gene insertion sequence.

  The production method according to the present invention preferably further includes a step of destroying the laminin α1 gene at the other locus among the pair of laminin α1 loci (laminin α1 gene disruption step). In the laminin α1 gene disruption step, a conventionally known conventional gene disruption method can be suitably used, and the specific method is not particularly limited. For example, in the examples described below, a method is used in which exogenous sequences are inserted into the laminin α1 gene and a part of the exon is deleted by homologous recombination.

  According to said method, the F9 embryonic tumor cell based on this invention can be produced simply and efficiently.

(III) Use of the present invention The field of use of the present invention is not particularly limited. Specifically, the method for producing an extracellular matrix using the F9 embryonic tumor cells, and the F9 embryonic tumor cells are used. Examples thereof include a method for screening a cell differentiation inducing factor, a differentiation inhibitory factor or an anticancer agent.

  Specifically, the extracellular matrix production method according to the present invention is not particularly limited as long as the above-described F9 embryonic tumor cells are used, and other specific configurations and materials are not particularly limited. According to the above method, a desired extracellular matrix can be efficiently produced using the above-mentioned F9 embryonic tumor cells.

  In addition, the screening method according to the present invention is not particularly limited as long as the above-described F9 embryonic tumor cells are used. Among the F9 embryonic tumor cells described above, those in which a marker gene is contained in the foreign gene insertion sequence are particularly preferable.

  Specifically, in the above-mentioned F9 embryonic tumor cells, the expression of the marker gene is subjected to the same expression control as that of the laminin α1 gene. Since the expression of the laminin α1 gene is used as a cell differentiation marker, the expression of a marker gene that undergoes the same expression control as the laminin α1 gene can be used as a cell differentiation marker.

  For example, when F9 +/− EGFP cells shown in Examples described later are induced to differentiate by treatment with retinoic acid and cAMP, the expression of laminin α1 gene is induced and the expression of EGFP gene is also induced. At this time, when a differentiation inhibiting factor is present, the expression of laminin α1 gene is not induced, and the expression of EGFP gene is not induced. Therefore, by confirming that the EGFP gene is not expressed by a fluorescence microscope or the like, it is possible to confirm the presence of a differentiation inhibiting factor. As described above, F9 embryonic tumor cells in which a marker gene such as EGFP is inserted into the foreign gene insertion sequence of the present invention can be used for screening for differentiation inhibitors.

  On the other hand, when differentiation is not induced by retinoic acid and cAMP treatment, the expression of laminin α1 gene is not induced, and the expression of EGFP gene is not induced. Here, for example, when a differentiation inducing factor is present, the expression of laminin α1 gene is induced and the expression of EGFP gene is also induced. Therefore, by confirming that the EGFP gene is expressed by a fluorescence microscope or the like, it is possible to confirm the presence of a differentiation inducing factor. Among known anticancer agents, those having a differentiation-inducing action are known, and therefore this screening system can be used for screening of anticancer agents using the differentiation-inducing action as an index.

  The differentiation inhibitor, differentiation inducer and anticancer agent may be an expression cDNA library. For example, the expression cDNA library is introduced into the F9 embryonic tumor cell according to the present invention, and the cells with enhanced fluorescence intensity are separated by flow cytomery, and then the expression cDNA library vector is recovered to obtain the differentiation-inducing factor. Can be identified. Similarly, after the expression cDNA library is introduced into the F9 embryonic tumor cells, differentiation is induced, and the cells whose fluorescence intensity does not increase are separated by flow cytometry, and then the expression cDNA library vector is recovered. By doing so, a differentiation inhibitory factor can be identified.

  Confirmation of the expression of the EGFP gene can be performed easily and inexpensively with a fluorescence microscope or the like. Therefore, F9 embryonic tumor cells (for example, F9 − / − EGFP cells or F9 +/− EGFP cells) in which the EGFP gene is inserted into the foreign gene insertion sequence are cell differentiation inducers from small to large scales, It can be suitably used for screening for differentiation inhibitors or anticancer agents.

  In addition, all of the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention will be described more specifically based on Examples and Comparative Examples and FIGS. 1 to 12, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

  In the present specification, “F9 − / − cell” means that one of a pair of laminin α1 genes is disrupted and a foreign gene insertion sequence is present on the other, and both laminin α1 genes are present. Shows missing cells. The “F9 +/− cell” is a cell in which a foreign gene insertion sequence is present in one of a pair of laminin α1 genes but the other remains as it is and only one of the laminin α1 genes is deleted. Indicates.

[Structure of gene targeting vector for obtaining homologous recombination F9 embryonic tumor cells]
FIG. 1 shows the structures of loxP targeting vector and lox2272-loxP targeting vector for obtaining homologous recombinant F9 embryonic tumor cells of the present invention.

  As shown in FIG. 1 (a), the lox2272-loxP targeting vector comprises a diphtheria toxin expression cassette (MC1-Diphteria toxin-PGK polyA), a lox2272 sequence (foreign gene insertion sequence), a G418 resistance cassette (G418r-PGK polyA), It has a loxp sequence (foreign gene insertion sequence).

  In the lox2272-loxP targeting vector of the present invention, a phosphoglucerokinase-derived polyadenylase is added to the G418 resistance gene so that it is controlled by the promoter of the mouse laminin α1 gene downstream of the transcription start origin of the first exon of the mouse laminin α1 gene. A G418 resistance cassette (G418r-PGK polyA) to which a translation sequence was added was incorporated, and the downstream sequence of the first exon of the mouse laminin α1 gene and a part of the first intron sequence were removed. The lox2272 sequence was inserted at the upstream end of the G418 resistance cassette and the loxP sequence was inserted at the downstream end (Araki K et al, Nucleic Acid Res (2002) 30: e103). A diphtheria toxin expression cassette is provided upstream of the promoter.

  In the lox2272-loxP targeting vector, the G418 resistance gene is expressed under the control of the laminin α1 gene promoter. Therefore, the G418 resistance gene functions as a marker for selecting cells in which the lox2272-loxP targeting vector has been incorporated. The diphtheria toxin expression cassette also suppresses non-specific integration of the lox2272-loxP targeting vector into the genome. When the lox2272-loxP targeting vector is integrated non-specifically into the genome, the diphtheria toxin A fragment is expressed in the cell, and the cell can be killed. In this example, the lox2272 sequence was inserted at the upstream end of the G418 resistance cassette and the loxP sequence was inserted at the downstream end. The region sandwiched between the lox2272 sequence and loxP sequence can be recombined with the region sandwiched by the lox2272 sequence and loxP sequence in other DNA fragments by CRE recombinase. That is, F9 embryonic tumor cells recombined with the laminin α1 locus by the lox2272-loxP targeting vector of the present invention are sandwiched between the lox2272 sequence and the loxP sequence by simultaneously introducing a gene conversion vector and a CRE recombinase expression vector described later. Recombination can occur between the defined regions. Therefore, the desired gene inserted into the region sandwiched between the lox2272 sequence and loxP sequence of the gene conversion vector is inserted downstream of the laminin α1 gene promoter by recombination. As a result, the desired gene can be expressed under the control of the laminin α1 gene promoter. In this example, the lox2272 sequence and loxP sequence were used as the foreign gene insertion sequence, and CRE recombinase was used as the enzyme that causes recombination. However, any sequence that causes recombination by CRE recombinase may be used, and is limited to the above sequence. is not. In addition, although CRE recombinase is used as an enzyme that causes recombination, it may be any enzyme that can cause recombination, and is not limited to the above enzyme. At this time, the foreign gene insertion sequence can be selected according to an enzyme capable of causing recombination.

  In addition, as shown in FIG. 1 (b), the loxP targeting vector comprises a diphtheria toxin expression cassette (MC1-Diphteria toxin-PGK polyA), a phosphoglucerokinase promoter, a G418 resistance cassette (G418r-PGK polyA) and a loxP sequence. Have.

  A G418 resistance cassette (G418r-), which is obtained by adding a phosphoglucerokinase-derived polyadenylation sequence to the G418 resistance gene downstream of the transcription start site of the first exon of the mouse laminin α1 gene so as to be controlled by the phosphoglucerokinase promoter. PGK polyA) was incorporated, and the downstream sequence of the first exon of the mouse laminin α1 gene and a part of the sequence of the first intron were removed. A loxP sequence was inserted into the upstream and downstream ends of the G418 resistance cassette. A diphtheria toxin expression cassette is provided upstream of the promoter.

  In the loxP targeting vector, the G418 resistance gene is expressed under the control of the phosphoglucerokinase promoter. Therefore, the G418 resistance gene functions as a marker for selecting cells in which the loxP targeting vector has been incorporated. The diphtheria toxin expression cassette also suppresses non-specific integration of the loxP targeting vector into the genome. When the loxP targeting vector is integrated non-specifically into the genome, the diphtheria toxin A fragment is expressed in the cell, and the cell can be killed.

[F9 having one destroyed laminin α1 locus and the other laminin α1 locus having a foreign gene insertion sequence for inserting a foreign gene so as to be expressed under the control of the promoter of the laminin α1 gene Establishment of embryonic tumor cells (F9 − / − cells)]
FIG. 2 shows a method for establishing F9 − / − cells.

  F9 embryonic tumor cells are karyotype 39WO, mouse-derived embryonic cancer cells having trisomy of chromosome 8 and monosomy of chromosome 14. In the F9 embryonic tumor cell, LAMA1, which is the locus of the laminin α1 gene, is present on chromosome 17.

  First, a loxP targeting vector linearized by restriction enzyme treatment was introduced into F9 embryonic tumor cells by electroporation, and cells into which the loxP targeting vector was incorporated were selected. Cells were selected using the expression of G418 resistance gene as an index. That is, clones that were resistant to geneticin were selected. Furthermore, by analyzing the selected clone by Southern blotting, a cell (# 54) in which the first exon of one LAMA1 locus was modified by homologous recombination was obtained.

  FIG. 3A shows a Southern blot of F9 embryonic tumor cells (wt) that have not undergone homologous recombination with F9 embryonic tumor cells (# 54) in which the first exon of the LAMA1 locus has been modified by homologous recombination. The analysis results are shown. FIG. 3B shows the arrangement of restriction enzymes near the laminin α1 gene in which the loxP targeting vector is incorporated.

  Genomic DNA purified from F9 embryonic tumor cells (# 54) and F9 embryonic tumor cells (wt) was enzymatically digested with SphI, NdeI, EcoRI + EcoRV, MluI + EcoRI or BamHI, respectively. The enzyme digestion product was analyzed by Southern blotting after electrophoresis. At this time, the LAMA1 probe represented by SEQ ID NO: 1 was used for the analysis of the enzyme digestion product by SphI, NdeI, EcoRI + EcoRV and MluI + EcoRI by the Southern blot method, and the analysis of the enzyme digestion product by BamHI was performed by the Southern blot method. The Neo probe indicated by 2 was used.

  In analysis of the enzyme digestion product by SphI, F9 embryonic tumor cells (# 54) showed bands of 3382 bp and 8075 bp, and F9 embryonic tumor cells (wt) showed a band of 8075 bp. In analysis of the enzyme digestion product by NdeI, F9 embryonic tumor cells (# 54) showed 7194 bp and 6772 bp bands, and F9 embryonic tumor cells (wt) showed 6772 bp bands. In the analysis of the enzyme digestion product by EcoRI + EcoRV, F9 embryonic tumor cells (# 54) showed 2927 bp and 9550 bp bands, and F9 embryonic tumor cells (wt) showed 9550 bp bands. In the analysis of the enzyme digestion product by MluI + EcoRI, F9 embryonic tumor cells (# 54) showed 5.2 kbp and 3.6 kbp bands, and F9 embryonic tumor cells (wt) showed a 3.6 kbp band. In the analysis of the enzyme digestion product by BamHI, # 54 showed a band of 7.8 kbp. As described above, F9 embryonic tumor cells (# 54) in which the first exon of one LAMA1 locus was modified by homologous recombination were selected.

  Next, CRE recombinase was transiently introduced into F9 embryonic tumor cells (# 54) in which the first exon of the LAMA1 locus was modified, and single cell clones were obtained by the limiting dilution method. At this time, expression of CRE recombinase causes recombination between loxP sequences, and F9 embryonic tumor cells lacking the G418 resistance cassette are obtained. Therefore, in order to select F9 embryonic tumor cells lacking the G418 resistance cassette, those showing the G418 sensitivity among the single cell clones were selected. In this clone, as shown in the upper right of FIG. 2, one copy of the laminin α1 locus is destroyed among the two copies of the laminin α1 locus.

  Furthermore, after introducing the linearized lox2272-loxP targeting vector into the clone by electroporation, the clone was again selected by geneticin. In this clone, the lox2272-loxP targeting vector undergoes homologous recombination with an unbroken copy of the laminin α1 locus. As a result, a clone in which both copies of the laminin α1 locus are destroyed is obtained. The selected clone is analyzed by Southern blotting to destroy one of the endogenous laminin α1 gene loci and to express the exogenous gene at the other laminin α1 gene locus under the control of the laminin α1 gene promoter. F9 embryonic tumor cells (F9 − / − cells) having an exogenous gene insertion sequence for insertion of F9 were obtained.

  Since the F9 − / − cell of the present invention has a foreign gene insertion sequence for inserting a foreign gene so as to be expressed under the promoter control of the laminin α1 gene, the desired gene can be controlled by the promoter of the laminin α1 gene. It can be expressed below. The promoter of laminin α1 gene is positively controlled when F9 embryonic tumor cells are induced to differentiate by treatment with retinoic acid and cAMP. Therefore, the F9 − / − cell of the present invention in which a desired gene is inserted into the foreign gene insertion sequence has low expression of the desired gene before differentiation induction by retinoic acid and cAMP treatment. After induction, the expression of the desired gene is highly induced. That is, the expression timing of a desired gene can be controlled.

  Moreover, the F9 − / − cells before differentiation induction by treatment with retinoic acid and cAMP, etc. rapidly proliferate. For example, when the gene inserted into the foreign gene insertion sequence is a gene having a cell growth inhibitory effect, the expression of the desired gene is suppressed to a low level unless differentiation is induced. Therefore, the F9 − / − cells before inducing differentiation proliferate rapidly. Therefore, in order to obtain a large number of cells expressing the desired gene, F9 − / − cells in which the desired gene is inserted into the foreign gene insertion sequence are cultured in large quantities before induction of differentiation, and then differentiation induction is performed. This makes it possible to obtain a large number of cells that express a desired gene. That is, if the F9 − / − cell of the present invention is used, a system for expressing a desired gene in large quantities can be easily scaled up.

  The F9 − / − cell has both laminin α1 loci disrupted. Therefore, since the F9 − / − cells do not express endogenous laminin α1 gene, endogenously expressed laminin β1 and laminin γ1 do not form a heterotrimeric protein with endogenous laminin α1. . On the other hand, when the α subunit is expressed exogenously, the F9 − / − cells form a heterotrimeric protein together with laminin β1 and laminin γ1 that are endogenously expressed. As a result, it becomes possible to preferentially express a desired laminin isoform derived from an α subunit that is expressed exogenously. The α subunit that is expressed exogenously at this time may be expressed by insertion into the foreign gene insertion sequence of F9 − / − cells, or may be expressed by another expression vector, and is particularly limited. is not.

  In addition, since both laminin α1 loci are disrupted in the F9 − / − cells, the expression of the α1 subunit is completely lost. Therefore, F9 − / − cells can be used as a laminin α1 knockout cell for functional analysis of the laminin α1 gene.

[Establishment of F9 embryonic tumor cells (F9 +/− cells) having a laminin α1 locus having a foreign gene insertion sequence for inserting a foreign gene so that the gene is expressed under the control of a laminin α1 promoter]
FIG. 4 shows a method for establishing F9 +/− cells.

  First, a lox2272-loxP targeting vector linearized by restriction enzyme treatment was introduced into F9 embryonic tumor cells by electroporation, and cells into which the lox2272-loxP targeting vector was incorporated were selected. Cells were selected using the expression of G418 resistance gene as an index. That is, clones that were resistant to geneticin were selected. Furthermore, the selected clone was analyzed by Southern blotting to obtain F9 +/− cells in which the first exon of one LAMA1 locus was modified by homologous recombination.

  Since the F9 +/− cell of the present invention has a foreign gene insertion sequence for inserting a foreign gene so as to be expressed under the promoter control of one laminin α1 gene, the desired gene is the promoter of the laminin α1 gene. It can be expressed under control. The promoter of laminin α1 gene is positively controlled when F9 embryonic tumor cells are induced to differentiate by treatment with retinoic acid and cAMP. Therefore, the F9 +/− cell of the present invention in which the desired gene is inserted into the foreign gene insertion sequence has a low expression of the desired gene before differentiation induction by retinoic acid and cAMP treatment, etc. After that, the expression of the desired gene is highly induced. That is, the expression timing of a desired gene can be controlled.

  In addition, the F9 +/− cells before differentiation induction by treatment with retinoic acid and cAMP, etc. proliferate rapidly. For example, when the gene inserted into the foreign gene insertion sequence is a gene having a cell growth inhibitory effect, the expression of the desired gene is suppressed to a low level unless differentiation is induced. Therefore, the F9 +/− cells before differentiation induction proliferate rapidly. Therefore, in order to obtain a large amount of cells expressing the desired gene, F9 +/− cells in which the desired gene is inserted into the foreign gene insertion sequence are cultured in large quantities before induction of differentiation, and then differentiation is induced. It becomes possible to obtain a large number of cells expressing a desired gene. That is, if the F9 +/− cell of the present invention is used, a system for expressing a desired gene in large quantities can be easily scaled up.

  At this time, the F9 +/− cell expresses an endogenous laminin α1 gene from one laminin α1 locus, and expresses a desired gene from the other laminin α1 locus. Therefore, laminin β1 and laminin γ1 that are endogenously expressed in the F9 +/− cell form a heterotrimeric protein with laminin α1 that is endogenously expressed, and heterotrimeric with the α subunit that is exogenously expressed. Form body proteins. As a result, the F9 +/− cell of the present invention can simultaneously express laminin-1 and a desired laminin isoform derived from an exogenously expressed α subunit.

[Structure of gene conversion plasmid]
As shown in FIGS. 2 and 4, F9 − / − cells and F9 +/− cells have a G418 resistance cassette flanked by lox2272 and loxP sequences at one laminin α1 locus. By simultaneously expressing in the F9 − / − and F9 +/− cells any DNA fragment (gene conversion plasmid) having a lox2272 sequence upstream and a loxP sequence downstream and a CRE recombinase expression plasmid, It becomes possible to convert a G418 resistance cassette sandwiched between lox2272 sequence and loxP sequence in a cell into an arbitrary DNA fragment sandwiched between lox2272 sequence and loxP sequence.

  FIG. 5 shows the structure of the gene conversion plasmid.

  The gene conversion plasmid has a lox2272 sequence, a pCINeo plasmid (Promega) derived intron sequence, a multicloning site, bovine growth hormone polyA, a puromycin resistance cassette (PGK promoter-Puromycin-resistance-PGK polyA), and a loxP sequence.

  The multicloning site in the gene conversion plasmid is a sequence containing one or more kinds of restriction enzyme sequences so that any gene can be inserted, but any gene can be inserted as long as it can be inserted. There is no particular limitation. By inserting the cDNA of an arbitrary foreign gene into the multicloning site and expressing the gene conversion plasmid and the CRE recombinase expression plasmid simultaneously in F9 − / − cells or F9 +/− cells, the intracellular lox2272 sequence and It becomes possible to convert a G418 resistance cassette sandwiched between loxP sequences into a DNA fragment sandwiched between lox2272 and loxP sequences in a gene conversion plasmid. As a result, the cDNA of the foreign gene is inserted into one laminin α1 locus in the F9 − / − cell or the F9 +/− cell. Since the foreign gene is inserted downstream of the laminin α1 gene promoter, the foreign gene is expressed under the control of the laminin α1 gene promoter. Therefore, when a laminin α1, α2, α3, α4, α5 gene or the like is inserted as a foreign gene, laminin derived from the foreign α subunit is expressed in cooperation with other endogenous basement membrane constituent protein genes. The As a result, a basement membrane-like extracellular matrix can be formed cooperatively with the endogenous basement membrane constituent protein.

  In this example, the lox2272 sequence and loxP sequence were used as the foreign gene insertion sequence, and CRE recombinase was used as the enzyme that causes recombination. However, any sequence that causes recombination by CRE recombinase may be used, and is limited to the above sequence. is not. Further, although CRE recombinase is used as an enzyme that causes recombination, it may be any enzyme that can cause recombination, and is not limited to the above enzyme. At this time, the foreign gene insertion sequence can be selected according to an enzyme capable of causing recombination.

  Further, the puromycin resistance cassette of the gene conversion plasmid of the present invention has a structure in which a phosphoglucerokinase-derived promoter sequence is added upstream of a puromycin resistance gene to which a phosphoglucerokinase-derived polyadenylation sequence is added. In F9 − / − cells or F9 +/− cells that have undergone gene conversion with the gene conversion vector, the G418 resistance cassette is removed and the puromycin resistance cassette is inserted. Therefore, since the genetically transformed cells are puromycin resistant and at the same time G418 sensitive, cells transformed with high efficiency can be selected using these drug resistances as indicators.

[Method for Establishing Homologous Recombinant F9 Embryonic Tumor Cells (F9 +/− EGFP Cells) and Use of the Cells]
FIG. 6 shows a method for establishing F9 +/− EGFP cells.

  Here, F9 +/− EGFP cells refer to F9 embryonic tumor cells in which the EGFP gene is inserted into the foreign gene insertion sequence of the F9 +/− cells.

  A green jellyfish-derived green fluorescent protein (EGFP: Clontech) cDNA was incorporated into the multicloning site of the gene conversion vector. This gene conversion vector was introduced into F9 +/− cells together with a CRE recombinase expression plasmid. The G418 resistance cassette flanked by lox2272 and loxP sequences in F9 +/− cells is converted into a DNA fragment containing the EGFP gene and puromycin resistance cassette flanked by lox2272 and loxP sequences in the gene conversion plasmid. Therefore, in order to select F9 +/− EGFP cells in which site-specific recombination occurred, first a puromycin resistant clone was selected, and then a clone showing G418 sensitivity was selected from the obtained clone. Thereafter, the selected cells were analyzed by Southern blotting to obtain F9 +/− EGFP cells in which site-specific recombination had occurred.

  In F9 +/− EGFP cells, since EGFP expression is controlled by the laminin α1 gene promoter, EGFP is expected to be expressed in the same manner as the LAMA1 gene. The laminin α1 gene is expressed at a very low level in undifferentiated F9 embryonic tumor cells, but is expressed at a high level when F9 embryonic tumor cells are differentiated into wall-side endoderm-like cells by retinoic acid and cAMP. The Therefore, after inducing differentiation of F9 +/− EGFP cells with retinoic acid and cAMP, the expression level of EGFP protein was confirmed by a fluorescence microscope, and the expression level of EGFP mRNA was confirmed by a real-time PCR method.

  FIG. 7 (a) is a diagram showing the form of F9 +/− EGFP cells and the expression level of EGFP protein 48 hours after differentiation induction with retinoic acid and cAMP and before differentiation induction, as observed by a fluorescence microscope. is there.

F9 +/− EGFP cells were plated on a glass slide coated with fibronectin so that the cell density was 1 × 10 ⁴ cells / cm ² , and cultured for 96 hours in DMEM medium containing 10% FBS. . At this time, retinoic acid and cAMP were added to the DMEM medium so that the final concentrations were 1 μM and 1 mM, respectively. After 96 hours of culture, F9 +/− EGFP cells were observed with a Pascal LSM5 confocal laser microscope.

  As shown in FIG. 7 (a), before F9 +/− EGFP cells are induced to differentiate, the cells do not show a differentiated form and the expression level of the EGFP protein is very low. On the other hand, 96 hours after the differentiation induction treatment, the cells show a differentiated form and the expression level of EGFP protein increases. Therefore, it was confirmed that the expression of the EGFP gene is controlled by the promoter of the laminin α1 gene, and that the expression pattern of the laminin α1 gene can be known by knowing the expression pattern of the EGFP gene.

  FIG. 7 (b) shows the endogenous laminin α1 mRNA (MLA) and endogenous laminin γ1 mRNA (MLC) of F9 +/− EGFP cells 96 hours after differentiation induction treatment with retinoic acid and cAMP quantified by real-time PCR. It is the figure which showed the expression level of EGFP mRNA (egfp).

F9 +/− EGFP cells were seeded on a gelatin-coated culture dish at a density of 1 × 10 ⁴ cells / cm ² and cultured in DMEM medium containing 10% FBS for 96 hours. . At this time, retinoic acid and cAMP were added to the DMEM medium so that the final concentrations were 1 μM and 1 mM, respectively. After 96 hours of culture, RNA was extracted from the cells using RNAeasy kit (Qiagen). CDNA was synthesized from 1 μg of total RNA using SuperScript II (Invitrogen) and random primers. Real-time PCR was performed using a QuantTect Cyber SYBR green PCR kit (Qiagen) and a Smart cycler (Cepheid) using 1/50 of the synthesized cDNA. At this time, the PCR reaction conditions were a pre-denaturation treatment at 95 ° C. for 900 seconds, a denaturation reaction at 95 ° C. for 10 seconds, an annealing reaction at 60 ° C. for 30 seconds, and an extension reaction at 72 ° C. for 60 seconds. For 45 cycles. The detected expression level of each gene was corrected based on the expression level of GAPDH (Glyseraldehyde 3-phosphate dehydrogenase) gene.

  In addition, the following primers were used for real-time PCR.

  To detect laminin α1, MLA forward primer: 5′-CTACCACAGCCTGAACTGTG-3 ′ (SEQ ID NO: 3) and MLA reverse primer: 5′-TGTATTCTGTCTTGACTGTGTG-3 ′ (SEQ ID NO: 4) were used. In order to detect laminin γ1, an MLC forward primer: 5′-TGTACCATGCCTGGTCACGT-3 ′ (SEQ ID NO: 5) and an MLC reverse primer: 5′-TGGCAATCTCCGAGATGGAG-3 ′ (SEQ ID NO: 6) were used. In order to detect EGFP, egfp forward primer: 5'-GGCGATGCCACCTACGGCAAG-3 '(SEQ ID NO: 7) and egfp reverse primer: 5'-GTTGCCGTCCTCCTTGAAGTCG-3' (SEQ ID NO: 8) were used. In order to detect GAPDH, a gapdh forward primer: 5'-GCAAAGTGGAGATTGTTGCCAT-3 '(SEQ ID NO: 9) and a gapdh reverse primer: 5'-GATGCAGGGATGATGTTCTGG-3' (SEQ ID NO: 10) were used.

  As shown in FIG. 7 (b), the amount of mRNA of laminin α1 and laminin γ1 expressed endogenously before the differentiation induction of F9 +/− EGFP cells is kept low. Similarly, the amount of EGFP mRNA to be expressed exogenously is also kept low. On the other hand, when F9 +/− EGFP cells were induced to differentiate, it was confirmed that the amounts of laminin α1, laminin γ1 and EGFP mRNA increased in parallel. Therefore, it was confirmed that the expression of the EGFP gene is controlled by the promoter of the laminin α1 gene, and that the expression pattern of the laminin α1 gene can be known by knowing the expression pattern of the EGFP gene.

  In the F9 +/− EGFP cells of the present invention, the EGFP gene is used as a marker gene. As a result, the expression of the EGFP gene is controlled by the promoter of the laminin α1 gene. Therefore, the EGFP gene is expressed in the same manner as the laminin α1 gene, and the expression pattern of the laminin α1 gene can be known by knowing the expression pattern of the EGFP gene.

  At this time, the expression of the EGFP gene can be confirmed simply and inexpensively with a fluorescence microscope or the like. Since it is possible to confirm the expression simply and inexpensively, it is possible to know whether or not the laminin α1 gene promoter has been activated in a large number of cell samples.

[Method for establishing homologous recombinant F9 embryonic tumor cells (F9 +/− LAMA5 cells)]
FIG. 8 shows a method for establishing F9 +/− LAMA5 cells.

  Here, the F9 +/− LAMA5 cell refers to an F9 embryonic tumor cell in which the laminin α5 gene is inserted into the foreign gene insertion sequence of the F9 +/− cell.

  Laminin α5 cDNA was incorporated into the multiple cloning site of the gene conversion vector. This gene conversion vector was introduced into F9 +/− cells together with a CRE recombinase expression plasmid. The G418 resistance cassette flanked by lox2272 and loxP sequences in F9 +/− cells is converted into a DNA fragment containing the laminin α5 gene and the puromycin resistance cassette flanked by lox2272 and loxP sequences in the gene conversion plasmid. . Therefore, in order to select F9 +/− LAMA5 cells in which site-specific recombination occurred, a puromycin resistant clone was first selected, and then a clone showing G418 sensitivity was selected from the obtained clone. Thereafter, the selected cells were analyzed by Southern blotting to obtain F9 +/− LAMA5 cells in which site-specific recombination had occurred.

The cells secrete laminin-1 (α1β1γ1) containing an α1 subunit expressed endogenously and laminin-10 (α5β1γ1) containing an α5 subunit expressed exogenously by differentiation induction.
In the F9 +/− cell of the present invention, the laminin α1, α2, α3, α4 or α5 gene may be inserted into the foreign gene insertion sequence. As a result, it becomes possible to induce the expression of the laminin α1, α2, α3, α4 or α5 gene by inducing differentiation of the F9 +/− cells by treatment with retinoic acid and cAMP. At this time, the F9 +/− cell expresses an endogenous laminin α1 gene from one laminin α1 locus, and laminin α1, α2, α3, α4 inserted into the foreign gene insertion sequence from the other laminin α1 locus. Alternatively, the α5 gene is expressed.

  Laminin β1 and laminin γ1 expressed endogenously in the F9 +/− cell form a heterotrimeric protein with laminin α1 expressed endogenously, and laminin α1, α2, α3, α4 expressed exogenously, or Heterotrimeric protein is also formed with α5. As a result, the desired laminin isoform and laminin-1 can be expressed simultaneously. That is, in F9 +/− cells genetically transformed with laminin α1, laminin-1 in F9 +/− cells genetically transformed with laminin α2, in F9 +/− cells genetically transformed with laminin-2 and laminin-1, laminin α3. Laminin-6 and laminin-1, laminin-8 and laminin-1 in F9 +/− cells transformed with laminin α4, laminin-10 and laminin-1 in F9 +/− cells transformed with laminin α5 are expressed. It becomes possible. As a result, a basement membrane-like structure containing the desired laminin isoform and laminin-1 can be obtained.

  In addition, since the laminin isoform exogenously expressed by the F9 +/− cell of the present invention is secreted to form a basement membrane-like structure, the exogenously expressed laminin isoform can be easily purified. Is possible.

[Method for establishing homologous recombinant F9 embryonic tumor cells (F9 − / − LAMA5 cells)]
FIG. 9 shows a method for establishing F9 − / − LAMA5 cells.

  Here, the F9 − / − LAMA5 cell refers to an F9 embryonic tumor cell in which the laminin α5 gene is inserted into the foreign gene insertion sequence of the F9 − / − cell.

  Laminin α5 cDNA was incorporated into the multiple cloning site of the gene conversion vector. This gene conversion vector was introduced into F9 − / − cells together with a CRE recombinase expression plasmid. The G418 resistance cassette flanked by lox2272 and loxP sequences in F9 − / − cells is converted into a DNA fragment containing the laminin α5 gene and the puromycin resistance cassette flanked by lox2272 and loxP sequences in the gene conversion plasmid. The Therefore, in order to select F9 − / − LAMA5 cells in which site-specific recombination occurred, first a puromycin resistant clone was selected, and then a clone showing G418 sensitivity was selected from the obtained clone. Thereafter, the selected cells were analyzed by Southern blotting to obtain F9 − / − LAMA5 cells in which site-specific recombination had occurred.

  Since no endogenously expressed α1 subunit is expressed in this cell, the endogenously expressed β1 subunit and γ1 subunit form a heterotrimeric protein with the exogenously expressed α5 subunit. . As a result, most of the secreted laminin is laminin-10 containing an exogenous α5 subunit. After differentiation induction of the cells thus obtained, the culture solution was analyzed by Western blotting using an anti-laminin α5 antibody, and the amount of laminin-10 secreted was estimated.

  The analysis result of the secreted laminin-10 by Western blotting is shown in FIG.

F9 cells, F9 − / − cells and F9 − / − LAMA5 cells are plated on gelatin-coated culture dishes to a density of 1 × 10 ⁴ cells / cm ² and 10% FBS is added. The culture was performed for 96 hours in the DMEM medium. At this time, retinoic acid and cAMP were added to the DMEM medium so that the final concentrations were 1 μM and 1 mM, respectively. After 96 hours of culture, the medium was collected from the culture dish. After 10 μl of the collected medium was electrophoresed under non-reducing conditions, Western blotting analysis was performed using an anti-laminin α5 antibody. At this time, F9 cells, F9 − / − cells, and F9 − / − LAMA5 cells use anti-laminin α5-specific monoclonal antibodies (see Non-Patent Document 2) for one type, one type, and four types of cell lines, respectively. Western blotting analysis was performed. In addition, in order to quantify laminin α5 secreted into the medium, 500 ng of recombinant laminin-10 was also electrophoresed, and Western blotting analysis was performed using anti-laminin α5 antibody. In FIG. Laminin-10, ie laminin β5 and laminin γ1, which formed a trimer with laminin β1, and a low molecular weight band indicates a monomer of laminin α5. As shown in FIG. 10, the expression of laminin-10 cannot be confirmed in F9 cells and F9 − / − cells both before and after differentiation induction. On the other hand, four lines of F9 − / − LAMA5 cells were shown to secrete laminin-10 into the medium after differentiation induction. At this time, by comparing the band intensity of laminin-10 of F9 − / − LAMA5 cells with the band intensity of 500 ng recombinant laminin-10 in Western blotting analysis, the concentration of laminin-10 in the medium was at least 10 ng. / Μl.
In the F9 − / − cell of the present invention, the laminin α1, α2, α3, α4 or α5 gene may be inserted into the foreign gene insertion sequence. As a result, it becomes possible to induce the expression of laminin α1, α2, α3, α4 or α5 gene by inducing differentiation of the F9 − / − cells by treatment with retinoic acid and cAMP. At this time, both laminin α1 loci are disrupted in the F9 − / − cells. Therefore, since the F9 − / − cell does not express the endogenous laminin α1 gene, the endogenously expressed laminin β1 and laminin γ1 of the F9 − / − cell are endogenous laminin α1 and heterotrimer. There is no protein formation. On the other hand, when laminin α1, α2, α3, α4 or α5 is expressed exogenously, the exogenously expressed laminin α chain forms a heterotrimeric protein together with endogenously expressed laminin β1 and laminin γ1. To do. As a result, the desired laminin isoform can be preferentially expressed. That is, laminin-1 in F9 − / − cells genetically transformed with laminin α1, laminin-2 in F9 − / − cells genetically transformed with laminin α2, and laminin in F9 − / − cells genetically transformed with laminin α3. -6, Laminin-8 can be preferentially expressed in F9 − / − cells genetically transformed with laminin α4, and Laminin-10 can be preferentially expressed in F9 − / − cells genetically transformed with laminin α5. As a result, a basement membrane-like structure containing a desired laminin isoform can be obtained.

  In addition, since the laminin isoform expressed exogenously in the F9 − / − cells of the present invention is secreted to form a basement membrane-like structure, the exogenously expressed laminin isoform can be easily purified. Is possible.

[Method for producing basement membrane-like structure containing laminin-1 and laminin-10 or basement membrane-like structure containing laminin-10 and not laminin-1 using F9 +/− LAMA5 cells and F9 − / − LAMA5 cells ]
Two glass slides coated with fibronectin so that each of F9 cells, F9 +/− LAMA5 cells, F9 − / − LAMA5 cells and F9 − / − cells has a density of 1 × 10 ⁴ cells / cm ^2. The cells were plated on top and cultured in DMEM medium containing 10% FBS for 96 hours. At this time, retinoic acid and cAMP were added to the DMEM medium so that the final concentrations were 1 μM and 1 mM, respectively. After culturing for 96 hours, the cells induced to differentiate into mural endoderm-like cells were fixed with 4% paraformaldehyde. Cells fixed on one glass slide were removed by lysing the cells with a solution containing 1% Triton X-100 and 20 mM NH ₄ OH. Thereafter, laminin α1 protein, laminin α5 protein and type IV collagen protein present on the cell membrane or in the basement membrane-like structure were visualized by fluorescent immunohistochemistry. As a primary antibody, rat anti-laminin α1 monoclonal antibody (Sanzen N et al. Unpublished), mouse anti-laminin α5 monoclonal antibody (see Non-Patent Document 2) or rabbit anti-type IV collagen polyclonal antibody (ELS) After the reaction, each was reacted with a FITC-labeled anti-rat IgG antibody (American Qualex), a FITC-labeled anti-mouse IgG antibody (American Qualex) or a Rhodamine-labeled anti-rabbit IgG antibody (BIOSOURCE) as a secondary antibody. . After the encapsulation treatment, the sample was observed with a Pascal LSM5 confocal laser microscope.

  FIG. 11 shows the results of analyzing the deposition of basement membrane constituents by F9 cells, F9 +/− LAMA5 cells, F9 − / − LAMA5 cells, and F9 − / − cells by a fluorescent immunohistochemical method. In FIG. 11, laminin α1 and laminin α5 are visualized in green, and type IV collagen is visualized in red.

  As shown in FIG. 11 (a), F9 cells and F9 +/− LAMA5 cells expressing endogenous laminin α1 deposit a basement membrane-like structure containing laminin-1 (α1β1γ1). On the other hand, F9 − / − LAMA5 cells and F9 − / − cells that do not express laminin α1 do not deposit a basement membrane-like structure containing laminin-1 (α1β1γ1). In addition, as shown in FIG. 11 (b), F9 cells and F9 − / − cells into which the laminin α5 gene has not been introduced do not deposit a structure containing laminin-10 (α5β1γ1). On the other hand, F9 +/− LAMA5 cells and F9 − / − LAMA5 cells into which a laminin α5 gene has been introduced deposit a structure containing laminin-10 (α5β1γ1).

  As described above, F9 +/− LAMA5 cells of the present invention express laminin-1 and laminin-10, and expressed laminin-1 and laminin-10 are secreted and deposited as a basement membrane-like structure. The F9 − / − LAMA5 cells of the present invention express laminin-10, and the expressed laminin-10 is secreted and deposited as a basement membrane-like structure. Accordingly, the F9 embryonic tumor cell of the present invention is cultured on a structure coated with gelatin, fibronectin, collagen, or the like, and induced to differentiate, whereby a basement membrane-like structure containing a desired protein on the structure is obtained. It becomes possible to deposit things. For example, if the F9 embryonic tumor cell of the present invention is cultured on a sponge made of collagen and induced to differentiate, a three-dimensional structure covered with a basement membrane-like substrate can be produced.

It is also possible to easily obtain a cell-free basement membrane-like structure by dissolving and removing cell components with phosphate buffered saline containing 0.5% Triton X-100 and 20 mM NH ₄ OH.

[F9 − / − Purification of Laminin-10 from LAMA5 Cell Culture Supernatant and Use of Purified Laminin-10]
F9 − / − LAMA5 cells were cultured on 20 100 mm culture dishes coated with gelatin. Each culture dish was seeded with 1 × 10 ⁶ cells and cultured in DMEM medium containing 10% FBS. At this time, 10 ml of DMEM medium was added to each culture dish, and retinoic acid and cAMP were further added to a final concentration of 1 μM and 1 mM, respectively. The culture medium of each culture dish was replaced with fresh DMEM medium 72 hours after the start of culture. The culture medium in each culture dish was collected 120 hours after the start of the culture, and 10 ml of the new DMEM medium was added to each culture dish and further cultured. 168 hours after the start of the culture, the medium in each culture dish was collected again, and 10 ml of the new DMEM medium was added to each culture dish and further cultured. And all the culture media were collect | recovered 240 hours after culture | cultivation start. As described above, 600 ml of the medium was collected.

  The collected 600 ml of the culture solution was purified using a column on which an anti-laminin α5 monoclonal antibody was immobilized. Approximately 600 μg of laminin-10 was purified in a single column pass.

  Laminin-10 produced by F9 − / − LAMA5 cells and F9 +/− LAMA5 has an α subunit derived from human and β and γ subunits derived from mouse. Laminin is a trimeric protein consisting of three subunits that undergo complex sugar modifications. Therefore, whether purified laminin has physiological activity is an important issue. Therefore, the physiological activity of laminin-10 purified from the F9 − / − LAMA5 cell culture supernatant was measured using cell spreading activity as an index.

96-well plates (Nunc) contain 0nM, 0.625nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM or 40nM (32μg / ml) EHS tumor-derived laminin (Sigma) or purified laminin-10 PBS was added in an amount of 50 μl per well, and coating treatment was performed at 4 ° C. for 12 hours. After the coating treatment, the 96-well plate was blocked with phosphate buffered saline containing 2% bovine serum albumin. HT1080 human fibrosarcoma cells are suspended in DMEM medium containing 0.5% bovine serum albumin to a concentration of 3 × 10 ⁵ cells / ml, 100 μl of this suspension is added to the 96-well plate, and CO ₂ Incubated for 30 minutes in incubator. Cells that adhered to the 96-well plate during the 30-minute incubation were fixed with an aqueous phosphate buffer solution containing 3.7% formalin. Thereafter, the cells were observed by staining with Diff quik reagent (see Hayashi Y et al, Biochem Biophys Res Commun 2002, 299: 498-504).

  As a result, in laminin-10, when the culture dish was coated at a concentration of 2.5 nM or more, HT1080 cells showed cell adhesion activity, and when the culture dish was coated at a concentration of 5 nM or more, HT1080 cells were strong cells. Extensive activity was demonstrated. On the other hand, in the case of laminin derived from EHS tumor, HT1080 cells showed cell adhesion activity when the culture dish was coated at a concentration of 10 nM or more, but even when the culture dish was coated at a concentration of 40 nM, the concentration of 10 nM was increased. The cell spreading activity was weaker than when the culture dish was coated with laminin-10.

  Therefore, it became clear that laminin-10 purified from the culture supernatant of F9 − / − LAMA5 cells of the present invention has physiological activity and its activity is higher than that of laminin purified from EHS tumor. .

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  As described above, F9 embryonic tumor cells according to the present invention can produce desired proteins including laminin isoforms in large quantities and at low cost. It can be used not only in the field of manufacturing various artificial tissues or organs represented by skin, liver and spleen, and parts thereof, but also in the fields of tissue engineering, developmental engineering and regenerative medicine. is there.

It is a schematic diagram showing the structure of a loxP targeting vector and a lox2272-loxP targeting vector for obtaining a laminin α1 gene modified F9 embryonic tumor cell line according to the present invention. It is a flowchart which shows the establishment method of F9-/-cell concerning this invention. It is a figure which shows the analysis result by Southern blotting of the laminin alpha1 gene modification F9 embryonic tumor cell line concerning this invention. It is a flowchart which shows the establishment method of F9 +/- cell concerning this invention. It is a schematic diagram which shows the structure of the gene conversion plasmid concerning this invention. It is a flowchart which shows the establishment method of F9 +/- EGFP cell concerning this invention. It is a figure which shows the change of the expression level of EGFP protein and EGFP mRNA accompanying the differentiation induction of the F9 +/- EGFP cell concerning this invention. It is a flowchart which shows the establishment method of F9 +/- LAMA5 cell concerning this invention. It is a flowchart which shows the establishment method of the F9-/-LAMA5 cell concerning this invention. It is a figure which shows the analysis result by the Western blotting method of laminin-10 which F9-/-LAMA5 cell concerning this invention secretes. It is a figure which shows the analysis result by the fluorescence immunohistochemical method of the basement membrane-like structure which the laminin alpha1 gene modification F9 embryonic tumor cell line concerning this invention secretes. It is a figure which shows the result of the cell adhesion assay using the laminin-10 refine | purified from the laminin alpha1 gene modification F9 embryonic tumor cell line concerning this invention.

Claims (11)

F9 embryonic tumor cells that can be used for at least protein production,
Of the pair of laminin α1 loci, one gene locus has a foreign gene insertion sequence for inserting a foreign gene so as to be expressed under the control of the laminin α1 gene promoter, instead of the laminin α1 gene. An F9 embryonic tumor cell, characterized in that it is provided.   The F9 embryonic tumor cell according to claim 1, wherein the laminin α1 gene is disrupted at the other gene locus.   The F9 embryonic tumor cell according to claim 1 or 2, wherein a gene encoding a protein constituting a basement membrane or a secreted protein is inserted into the foreign gene insertion sequence.   The F9 embryonic tumor cell according to claim 3, wherein the gene encoding the protein constituting the basement membrane or the secreted protein is a laminin α1, α2, α3, α4 or α5 gene.   The F9 embryonic tumor cell according to any one of claims 1 to 4, wherein the foreign gene insertion sequence includes a marker gene.   The F9 embryonic tumor cell according to claim 5, wherein the marker gene is a gene encoding a fluorescent protein, a luciferase gene, or a secreted alkaline phosphatase gene.   A method for producing an extracellular matrix using the F9 embryonic tumor cell according to any one of claims 1 to 6.   A method for screening for a cell differentiation-inducing factor, differentiation inhibitory factor or anticancer agent using the F9 embryonic tumor cell according to any one of claims 1 to 6. A method for producing F9 embryonic tumor cells that can be used at least for protein production,
Step of incorporating a foreign gene insertion sequence for inserting a foreign gene into one of the pair of laminin α1 gene loci so that the gene is expressed under the control of the laminin α1 gene promoter instead of the laminin gene A method for producing F9 embryonic tumor cells, comprising: The method for producing F9 embryonic tumor cells according to claim 9 , further comprising a step of inserting a gene that constitutes a basement membrane or a secretory protein into the foreign gene insertion sequence.   The method for producing F9 embryonic tumor cells according to claim 9 or 10, further comprising a step of destroying the laminin α1 gene at the other locus among the pair of laminin α1 loci.