WO2013183702A1 - Helper-dependent adenovirus vector, method for visualizing mature osteoblasts, and method for producing mature osteoblasts - Google Patents

Abstract

A helper-dependent adenovirus vector in which the first exon base sequence of an osteocalcin gene is substituted by a base sequence containing a reporter gene, and including at least part of the 5' upstream region of the first exon of the osteocalcin gene and at least part of the 3' downstream region of the first exon of the osteocalcin gene; and a method for visualizing and method for producing mature osteoblasts using this helper-dependent adenovirus vector.

Description

Helper-dependent adenovirus vector, method for visualizing mature osteoblasts, and method for producing mature osteoblasts

The present invention relates to a helper-dependent adenovirus vector, a method for visualizing mature osteoblasts and a method for visualizing mature osteoblasts using the helper-dependent adenovirus vector.

Osteoblasts are undifferentiated mesenchymal progenitor cells that are specialized for bone formation. The osteoblast is an organic extracellular matrix such as type I collagen (type I collagen), osteopontin, osteonectin, bone sialoprotein, and osteocalcin. It is secreted to form osteoid, which is a bone matrix (see, for example, Non-Patent Documents 1 and 2). Osteoid mineralization is induced by the enzymatic activity of alkaline phosphatase, a GPI-anchored protein expressed on the cell membrane of osteoblasts. The osteogenic ability of osteoblasts is controlled by systemic hormones, local growth factors, and cytokines. The transcription factors osterix (hereinafter sometimes referred to as “Osx”) and Runx2 have been shown to be essential for bone formation during embryonic development.

Osteocalcin is one of the most abundant non-collagenous proteins in the bone matrix, and the osteocalcin has a high affinity for hydroxyapatite via gamma carboxyglutamic acid (see, for example, Non-Patent Document 2). Osteocalcin knockout mice showed increased bone formation (see, for example, Non-Patent Document 3). Osteocalcin is the only gene that is specifically expressed in osteogenic cells in the osteogenic state, and the osteocalcin was identified in cubic cells on the bone surface of the bone marrow cavity. In primary osteoblasts prepared from bone tissue, the expression level of osteocalcin mRNA gradually increases in parallel with an increase in the number of bone-like nodules. The bone metabolism hormone 1α, 25 (OH) ₂ D ₃ (hereinafter sometimes referred to as “VD3”) is a vitamin D receptor regulatory element discovered in the 5 ′ upstream region of the rat and human osteocalcin genes. (Hereinafter, referred to as “VDREs”) up-regulates the expression of osteocalcin mRNA in rat and human osteoblasts (see, for example, Non-Patent Document 4). Therefore, in order to distinguish immature osteoblasts and other types of cells from mature osteoblasts, osteocalcin expression is widely used as a specific marker for mature osteoblasts.

The osteoblast has been conventionally separated from bone tissue by an enzyme digestion method or the like. However, those obtained by the above separation include various osteoblasts with different differentiation and activation states, progenitor cells thereof, etc., and lived mature osteoblasts in the osteogenic state. It is required to sort in the state.

As a useful technique for sorting a specific type of living cells from a mixed cell group, there is a method using a fluorescence-displayed cell sorting device (hereinafter sometimes referred to as “FACS”). . In order to sort live cells by FACS, a specific surface antigen is required. However, there is a problem that specific surface antigens for mature osteoblasts have not yet been identified. In addition, since osteocalcin is a secreted protein, mature osteoblasts cannot be separated by the FACS even using an anti-osteocalcin antibody, and a technique for sorting mature osteoblasts in a living state has not yet been developed. The current situation is not.

Therefore, there is a strong demand for rapid development of a technique that can efficiently sort mature osteoblasts in a living state.

This invention makes it a subject to solve the said conventional problems and to achieve the following objectives. That is, the present invention relates to a helper-dependent adenovirus vector that can visualize mature osteoblasts, a method for visualizing mature osteoblasts using the helper-dependent adenovirus vector, and a state in which mature osteoblasts are alive It is an object of the present invention to provide a method for producing mature osteoblasts that can be sorted by the above method.

Means for solving the problems are as follows. That is,
<1> The base sequence of the first exon of the osteocalcin gene is replaced with a base sequence containing a reporter gene,
At least part of the 5 ′ upstream region of the first exon of the osteocalcin gene;
A helper-dependent adenovirus vector comprising at least part of the 3 ′ downstream region of the first exon of the osteocalcin gene.
<2> Infecting osteoblasts with the helper-dependent adenovirus vector according to <1>,
A method for visualizing mature osteoblasts, comprising the step of detecting the expression of a reporter gene in the infected osteoblasts.
<3> Infecting osteoblasts with the helper-dependent adenovirus vector according to <1>,
Detecting the expression of a reporter gene in the infected osteoblasts;
A method for producing mature osteoblasts comprising the step of sorting cells in which expression of the reporter gene is detected.

According to the present invention, the above-mentioned problems in the prior art can be solved, and a helper-dependent adenovirus vector that can visualize mature osteoblasts, and a method for visualizing mature osteoblasts using the helper-dependent adenovirus vector And a method for producing mature osteoblasts capable of sorting mature osteoblasts in a living state.

FIG. 1 is a schematic explanatory diagram of an example of the helper-dependent adenovirus vector of the present invention produced in Production Example 1. FIG. 2A is a diagram showing the result of fluorescence microscope observation of Test Example 1. FIG. 2B is a diagram showing the results of FACS analysis of Test Example 1. FIG. FIG. 2C is a diagram showing the results of quantitative RT-PCR analysis of Venus gene in Test Example 1. FIG. 2D is a diagram showing the results of quantitative RT-PCR analysis of the endogenous osteocalcin gene in Test Example 1. FIG. 3A is a diagram illustrating a result of photographing using a 20 × objective lens in Test Example 2. FIG. 3B is a diagram illustrating a result of photographing using a 10 × objective lens in Test Example 2. FIG. 4A is a diagram showing the results of FACS analysis when the adenovirus vector obtained in Production Example 1 was used in Test Example 3. FIG. 4B is a diagram showing the results of FACS analysis when the adenovirus vector obtained in Comparative Production Example 1 was used in Test Example 3. FIG. 4C is a diagram showing the results of FACS analysis when no adenovirus vector was used in Test Example 3. FIG. 4D is a diagram showing the results of quantitative RT-PCR analysis in Test Example 3. FIG. 4E is a diagram showing the results of quantitative RT-PCR analysis in Test Example 3. FIG. 5A is a diagram showing a photographing result in Test Example 4. FIG. 5B is a diagram showing the results of FACS analysis when infecting the adenovirus vector obtained in Production Example 1 in Test Example 4. FIG. 5C is a diagram showing the results of FACS analysis when the adenovirus vector obtained in Comparative Production Example 2 was infected in Test Example 4. FIG. 5D is a diagram showing the results of FACS analysis when the adenovirus vector obtained in Comparative Production Example 3 was infected in Test Example 4. FIG. 5E is a diagram showing the results of FACS analysis in Test Example 4 when no adenovirus vector was infected. FIG. 6 is a schematic explanatory diagram of the E1 / E3-deficient adenovirus vector produced in Comparative Production Example 2.

(Helper-dependent adenovirus vector)
In the helper-dependent adenovirus vector of the present invention, the base sequence of the first exon of the osteocalcin gene is replaced with a base sequence containing a reporter gene, and at least one of the 5 ′ upstream region of the first exon of the osteocalcin gene. And at least a part of the 3 ′ downstream region of the first exon of the osteocalcin gene, and further includes other components as necessary.

<Osteocalcin gene>
The osteocalcin (also referred to as “BGLAP”) gene is a gene that is specifically expressed in osteogenic cells in the osteogenic state.
There is no restriction | limiting in particular as a seed | species of the said osteocalcin gene, According to the objective, it can select suitably, For example, a human, a mouse | mouth etc. are mentioned. Among these, human is preferable.
There is no restriction | limiting in particular as the acquisition method of the said osteocalcin gene, According to the objective, it can select suitably, For example, it can obtain from the BAC clone which has the genomic sequence of a human osteocalcin gene area | region.
Specific examples of the BAC clone include RP11-54H19 (BACPAC resources, Children's Hospital & Research Center at Oakland).

-5 'upstream region of the first exon-
The 5 ′ upstream region of the first exon is not particularly limited as long as it contains at least a part, and can be appropriately selected according to the purpose. A region exceeding 8 kb (kilobase pairs) is preferable, and a region from the translation start point of the first exon to 10.1 kb is more preferable. The more preferable region is advantageous in that the expression level of the reporter gene in mature osteoblasts can be increased, and mature osteoblasts can be more easily visualized and sorted.
The nucleotide sequence of the region from the translation start point of the first exon to 10.1 kb is represented by SEQ ID NO: 35 in humans.
When the 5 ′ upstream region is represented by “kb”, it is rounded up to the second decimal place. When the sequence represented by SEQ ID NO: 35 is expressed by “bp (base pair)”, it is 10,092 bp.

-3 'downstream region of the first exon-
The 3 ′ downstream region of the first exon is not particularly limited as long as it includes at least a part thereof, and can be appropriately selected according to the purpose. From the end of the first exon to 8.8 kb It is preferable that this region. The preferred region is advantageous in that the expression level of the reporter gene in mature osteoblasts can be increased, and mature osteoblasts can be more easily visualized and sorted.
The base sequence of the region from the end of the first exon to 8.8 kb is represented by SEQ ID NO: 36 in humans.
When the 3 ′ downstream region is represented by “kb”, it is rounded up to the second decimal place. When the sequence represented by SEQ ID NO: 36 is represented by “bp”, it is 8,758 bp.

<Base sequence including reporter gene>
The base sequence including the reporter gene includes at least a reporter gene, and further includes other base sequences as necessary.
The position of the base sequence containing the reporter gene is not particularly limited and may be appropriately selected depending on the intended purpose. However, at least a part of the 5 ′ upstream region of the first exon of the osteocalcin gene and the osteocalcin It is preferably located between at least part of the 3 ′ downstream region of the first exon of the gene.

-Reporter gene-
There is no restriction | limiting in particular as said reporter gene, Although it can select suitably according to the objective, A fluorescent protein gene is preferable.
There is no restriction | limiting in particular as said fluorescent protein, According to the objective, it can select suitably, For example, YFP, GFP, CFP, etc. are mentioned. Among these, Venus, which is a kind of GFP, is preferable in terms of the speed and intensity of fluorescence emission.
There is no restriction | limiting in particular as the acquisition method of the said reporter gene, According to the objective, it can select suitably, For example, it can obtain from the plasmid which has the said reporter gene.
Specific examples of the plasmid having the Venus gene include pCS2-Venus (RIKEN).

-Other base sequences-
The other base sequences are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, base sequences of antibiotic resistance genes, pA addition sequences, site-specific combinations Examples include replacement sequences and promoter sequences. The other base sequence preferably includes the base sequence of the antibiotic resistance gene.

The antibiotic resistance gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a neomycin resistance gene, a puromycin resistance gene, a blasticidin resistance gene, and a hygromycin resistance gene.

The helper-dependent adenovirus vector includes a terminal inversion sequence (ITR) at both ends and a packaging signal as necessary components to be amplified as a helper-dependent adenovirus and enclosed in a virus particle.

<Other configurations>
The other configuration is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a thymidine kinase (TK) gene sequence and a β-galactosidase gene sequence.

<Method for producing helper-dependent adenovirus vector>
The method for producing the helper-dependent adenovirus vector is not particularly limited, and can be produced by appropriately selecting a known method. The helper-dependent adenovirus vector of the present invention includes a plasmid embodiment, a virus particle Any of the embodiments are included.

The method for producing the helper-dependent adenovirus vector plasmid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the Red / ET recombination method based on homologous recombination in E. coli (“ Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci 2000; .
Hereinafter, an example of a method for producing the helper-dependent adenovirus vector plasmid of the present invention using the Red / ET recombination method will be described.

First, a eukaryotic promoter-prokaryotic promoter (hereinafter referred to as a eukaryotic promoter and a prokaryotic promoter) in which a site-specific recombination target sequence is arranged before and after the pA addition sequence of the reporter gene of a plasmid having a reporter gene. The promoter may be collectively referred to as “promoter”)-antibiotic regime gene-pA added sequence expression cassette (site-specific recombination target sequence-promoter-antibiotic resistance gene-pA-site-specific recombination target Sequence cassette) is inserted to create a plasmid with reporter gene-pA-site specific recombination target sequence-promoter-antibiotic resistance gene-pA-site specific recombination target sequence cassette.
The eukaryotic promoter is not particularly limited as long as it is a low-level constitutive expression promoter, and can be appropriately selected according to the purpose. For example, SV40 promoter, β-actin promoter, PGK (phosphoglycerate kinase) ) Promoters.
The prokaryotic promoter is used for clone selection when performing the Red / ET method, and examples thereof include a bla (β-lactamase) promoter and an EM7 promoter.
The site-specific recombination target sequence is for removing an antibiotic resistance gene cassette later and is not necessarily essential, but can be appropriately selected and used, for example, FRT sequence, loxP sequence, etc. It is done.

Next, the plasmid having the reporter gene-pA-site-specific recombination target sequence-promoter-antibiotic resistance gene-pA-site-specific recombination target sequence cassette is used as a template, and by PCR, the first exon of the osteocalcin gene is detected. A reporter gene-pA-site-specific recombination target sequence-promoter-antibiotic resistance gene-pA-site-specific recombination target sequence cassette with about 40 base pairs homologous arms homologous to the start codon region added on both sides Amplify.
The amplified cassette is inserted into the position of the start codon of the first exon of the osteocalcin gene encoded by the BAC clone by homologous recombination in E. coli.

Furthermore, the osteocalcin gene containing the introduced expression cassette by homologous recombination in E. coli using a linear cloning vector to which a homologous arm of about 40 base pairs homologous to the position of the target osteocalcin gene region is added by PCR amplification. Reclaim the area.

The osteocalcin gene region containing the recovered introduced expression cassette is subcloned into a helper-dependent adenovirus vector plasmid.
The helper-dependent adenovirus vector plasmid is not particularly limited and may be appropriately selected depending on the intended purpose. For example, pAMHDAdGT8-4 (“Aizawa E, Hirabayashi Y, Iwanaga Y et al. Efficient and Accumulated Recognition in Japan) in hESCs and hiPSCs Using Helper-dependent Adenoviral Vectors. Mol Ther 2011; 20 (2): 424-31.)), pCIHDAdGT8-3, and the like.

There is no restriction | limiting in particular as a manufacturing method of the virus particle of the said helper dependence type adenovirus vector, According to the objective, it can select suitably.
For example, when an FRT sequence is used as the site-specific recombination target sequence, 116 cells (Palmer D, Ng P. Improved system for helper-dependent adventor vector production. Mol.) That constantly expresses Cre recombinase. Ther 2003; 8 (5): 846-52.), The above-described helper-dependent adenovirus vector plasmid linearized is introduced, and further, a helper virus in which a packaging signal is sandwiched between two loxP sequences is infected. By doing so, virus particles can be produced.
Also, for example, when the loxP sequence is used as the site-specific recombination target sequence, the helper-dependent adenovirus vector plasmid linearized is introduced into 293 FLPe cells that constantly express the FLPe recombinase. Furthermore, virus particles can be produced by infecting a helper virus in which a packaging signal is sandwiched between two FRT sequences.
The plasmid introduction method is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a lipofection method.
The method for linearizing the plasmid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a method using a restriction enzyme.
The helper virus in which the packaging signal is sandwiched between two loxP sequences is not particularly limited and can be appropriately selected depending on the purpose. For example, AdNG163R-2 (Dr. Phillip Ng (Baylor College of Medicine) ), AdNG163 and the like.
The helper virus in which the packaging signal is sandwiched between two FRT sequences is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include FL helper.

As a preferred embodiment of the helper-dependent adenovirus vector of the present invention, a sequence of a region from the translation start point of the first exon of the osteocalcin gene to 10.1 kb 5 ′ upstream, a Venus gene sequence that is a reporter gene, 7. pA addition sequence, FRT sequence, PGK promoter sequence, EM7 promoter sequence, neomycin resistance gene sequence, pA addition sequence, FRT sequence, and 3 ′ downstream from the end of the first exon of the osteocalcin gene. An embodiment including an array of regions up to 8 kb in this order is preferable.

In the helper-dependent adenovirus vector of the present invention, the reporter gene is inserted into the osteocalcin gene region, so that the reporter gene is expressed under the control of the osteocalcin gene region. In the present invention, the osteocalcin region includes at least a part of the 5 ′ upstream region of the first exon and at least a part of the 3 ′ downstream region of the first exon, so that it is shown in a test example described later. In addition, it is considered that the reporter gene is expressed under the control of the osteocalcin gene region.
In the present invention, since a helper-dependent adenovirus vector is used, it can be applied to various cells and has an advantage of transient expression.
Therefore, it can be suitably used in a method for visualizing mature osteoblasts and a method for producing mature osteoblasts, which are in the state of forming bone, which will be described later. Moreover, it can be used suitably also for the study of transcriptional regulation.

(How to visualize mature osteoblasts)
The method for visualizing mature osteoblasts of the present invention includes at least an infection step and a detection step, and further includes other steps as necessary.

<Infectious process>
The infection step is a step of infecting osteoblasts with the helper-dependent adenovirus vector of the present invention.
The embodiment of the helper-dependent adenovirus vector used in the infection step is preferably a virus particle embodiment.

-Osteoblast-
There is no restriction | limiting in particular as said osteoblast seed | species, According to the objective, it can select suitably.
There is no restriction | limiting in particular as a preparation method of the said osteoblast, A well-known method can be selected suitably, For example, the method of isolate | separating from a bone tissue by an enzyme digestion method etc. is mentioned.
The osteoblast culture medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include αMEM containing 10% FCS containing 50 U / mL streptomycin and 50 μg / mL penicillin. It is done.
The culture conditions for the osteoblast are not particularly limited and may be appropriately selected depending on the intended purpose. Examples include culturing at 5% CO ₂ and 37 ° C., and changing the medium every 48 hours. It is done.
By the above culture, bone-like nodules are formed in primary osteoblasts in 1 to 2 weeks.

-infection-
The method for infecting osteoblasts with the helper-dependent adenovirus vector is not particularly limited, and a known method can be appropriately selected. For example, “Suzuki K, Mitsui K, Aizawa E et al. Highly efficient. Transgene gene expression and gene targeting in prime embryonic stem cells with helper-dependent adenovial vectors. Proc Natl AA.

The infection ratio (MOI) in the infection is not particularly limited and can be appropriately selected according to the purpose. However, when the physical titer is 1,000 to 10,000, the infection efficiency is 60% or more. It is preferable in that the cytotoxicity can be prevented while achieving it.

The method for adjusting the titer of the helper-dependent adenovirus vector is not particularly limited, and can be adjusted by appropriately selecting a known method.
The method for measuring the titer of the helper-dependent adenovirus vector is not particularly limited, and a known method can be appropriately selected. For example, the infectious titer is measured by X-gal staining, and genomic Southern hybridization is performed. (Palmer DJ, Ng P. Physical and infectious titers of heper-dependent adenorientor vectors: a method of direct compensator. 792-8.)).

<Detection process>
The detection step is a step of detecting the expression of the reporter gene in the osteoblasts infected in the infection step.

-detection-
The means for detecting the expression of the reporter gene is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include FACS (manufactured by BD Biosciences) and fluorescence microscope (manufactured by Olympus).
According to the visualization method of the present invention, mature osteoblasts that are osteogenic can be visualized by detecting the expression of a reporter gene.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably.

(Method for producing mature osteoblasts)
The method for producing mature osteoblasts of the present invention includes at least an infection step, a detection step, and a sorting step, and further includes other steps as necessary.

<Infectious process>
The infection step is a step of infecting osteoblasts with the helper-dependent adenovirus vector of the present invention, and can be performed in the same manner as the infection step in the above-described method for visualizing mature osteoblasts.

<Detection process>
The detection step is a step of detecting the expression of the reporter gene in the osteoblasts infected in the infection step, and can be performed in the same manner as the detection step in the above-described method for visualizing mature osteoblasts.

<Preparation process>
The sorting step is a step of sorting the cells in which the expression of the reporter gene is detected in the detection step.

-Preparation-
The means for sorting the cells in which the expression of the reporter gene is detected is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include FACS (manufactured by BD Biosciences).
According to the production method of the present invention, mature osteoblasts that are in a bone-forming state can be sorted in a living state.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably.

Hereinafter, production examples and test examples of the present invention will be described, but the present invention is not limited to these production examples and test examples.

(Cell culture)
Cell culture in each of the following production examples, comparative production examples, and test examples was performed as follows.
<116 cells>
116 cells (Palmer D, Ng P. Improved system for helper-dependent adenoviral vector production. Mol Ther 2003; 8 (5): 846-52. See Dr. Philly N. The cells were cultured in MEM (manufactured by Nacalai Tesque) containing% FCS (manufactured by MP Biomedicals).
The 116 cells are a human fetal kidney 293 cell line that expresses Cre recombinase.

<Human osteosarcoma MG-63 cells>
Human osteosarcoma MG-63 cells (Billiau A, Edy VG, Heremans H et al. Human interferon: mass production in a newly established cell line, MG-63. (Obtained from Tohoku University Medical Research Center for Aging Medicine) containing 1 mM sodium pyruvate (Sigma-Aldrich) and 1% non-essential amino acid (Sigma-Aldrich) containing 10% FCS Of MEM (manufactured by Nacalai Tesque).

<293A cells>
Adhesive 293 cell line 293A (obtained from Life Technologies) was cultured in DMEM (manufactured by Nacalai Tesque) containing 10% FCS.

(Production Example 1: Helper-dependent adenovirus vector-1)
A helper-dependent adenovirus vector-1 (hereinafter, sometimes referred to as “HDAd-hOC-Venus”, about 30 kb) is a BAC clone (RP11−) having the genomic sequence of the human osteocalcin (also referred to as “BGLAP”) gene region. Based on 54H19, BACPAC resources, Children's Hospital & Research Center at Oakland), the Red / ET recombination method based on homologous recombination in Escherichia coli (“Datsenko KA, Wanner BL. One-step inactivity”). chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci US A 2000; 97 (12): 6640-5. ”).

First, pCS2-Venus, an expression plasmid containing the fluorescent protein Venus, which is a reporter gene (obtained from Dr. Atsushi Miyawaki, RIKEN, “Nagai T, Ibata K, Park ES et al. A variant of the United States”). yeast-specific site-specific recombination at the NotI site (behind the pA-added sequence of the fluorescent protein Venus gene) in the effective material for cell-biological applications. See Nat Biotechnol 2002; 20 (1): 87-90. FRT sequence, which is the target sequence, is arranged before and after Inserted PGK promoter-EM7 promoter-neomycin resistance gene-pA added sequence expression cassette (FRT-PGK-EM7-neo-pA-FRT cassette) and inserted Venus-pA-FRT-PGK-EM7-neo-pA-FRT A plasmid with a cassette was obtained.

Next, the plasmid clone having the Venus-pA-FRT-PGK-EM7-neo-pA-FRT cassette was used as a template, and PCR was performed using primers (SEQ ID NOs: 1 and 2 in Table 1) having a 40-nucleotide target arm. Amplified was a Venus-pA-FRT-PGK-EM7-neo-pA-FRT cassette in which homologous arms of 40 base pairs each homologous to the osteocalcin gene start codon region were added on both sides. The amplified cassette was inserted at the position of the start codon of the first exon of the osteocalcin gene encoded by the BAC clone by homologous recombination in E. coli (see FIGS. 1A to 1C).

Furthermore, pBR322 plasmid vector ("Bolivar F, Rodriguez RL, Betlac MC et al. Construction and charac- terization of new cloning vehicles. II. A multipurposse. And 5 ′ upstream region 10.1 kb and 3 ′ downstream region 8.8 kb of the first exon of the osteocalcin gene prepared by PCR amplification using the primers of SEQ ID NOs: 3 and 4 in Table 1. Osteocalcin inheritance including the introduced expression cassette by homologous recombination in E. coli using a linearized plasmid vector fragment to which a homologous 40 base pair homologous arm is added Region (5 ′ upstream region 10.1 kb (see SEQ ID NO: 35 (10,092 bp))-Venus-pA-FRT-PGK-EM7-neo-pA-FRT-3 ′ downstream region 8.8 kb (SEQ ID NO: : 36 (8,758 bp)) was recovered.
The 5 ′ upstream region 10.1 kb includes Runx2, Vitamin D receptor binding sequence.

In the sequences in Table 1, italic letters indicate additional sequences that do not hybridize with the target DNA, and underlined portions indicate restriction enzyme recognition sites.

The osteocalcin gene region (21.4 kb in total) containing the recovered introduced expression cassette was transferred to a helper-dependent adenovirus vector plasmid, pAMHDAdGT8-4 (“Aizawa E, Hirabayashi Y, Iwanaga Y et al. Efficient Hocino Regenerative Hoc.). hESCs and hiPSCs Usage Helper-dependent Adenoviral Vectors. Mol Ther 2011; 20 (2): 424-31.)) (see D in FIG. 1).

The obtained helper-dependent adenovirus vector plasmid (hereinafter sometimes referred to as “pAMHDAdGT-hOC-Venus”) is linearized with the restriction enzyme PmeI, and the Cre recombinase derived from E. coli P1 phage is constitutively expressed. The cells were transiently introduced into 116 cells, which were 293 cells, by the lipofection method.
The cells are further infected with the helper virus AdNG163R-2 (obtained from Dr. Phillip Ng (available from Baylor College of Medicine)), which is not packaged in the 116 strain because the packaging signal is sandwiched between two loxP sequences. Thus, only the helper-dependent adenovirus vector was packaged in adenovirus (see FIG. 1, E, “Palmer D, Ng P. Improved system for helper-dependent adrenal vector production. Mol Ther 5 2003; ): 846-52.

In FIG. 1, “A” represents a 2.6 kb PCR fragment of the Venus-pA-FRT-PGK-EM7-neo-pA-FRT cassette amplified by using a primer with a 40 nucleotide homologous arm. . “ATG” indicates a translation initiation codon.
In FIG. 1, “B” indicates the human osteocalcin gene region in the BAC clone “RP11-54H19”. “VDRE” refers to a vitamin D receptor regulatory element.
In FIG. 1, “C” indicates a human osteocalcin gene region into which the Venus gene has been inserted by Red / ET homologous recombination.
In FIG. 1, “D” indicates a pAMHDAd-hOC-Venus vector constructed by subcloning a total of 21.4 kb of the sequence including the marker cassette into pAMHDAdGT8-4. “5 ′ upstream” indicates the 5 ′ upstream region of the first exon of the 10.1 kb human osteocalcin gene. “3 ′ downstream” indicates the 3 ′ downstream region of the first exon of the 8.8 kb human osteocalcin gene. “LacZ” indicates a β-galactosidase gene having a mouse cytomegalovirus promoter and SV40 pA. “TK” indicates “a thymidine kinase gene of a herpes simplex virus having a promoter and a pA signal in the opposite direction to the osteocalcin gene”. “Arrows at both ends” indicates the terminal inverted sequence (ITR) of the adenoviral vector.
In FIG. 1, “E” is a schematic view of a packaged state.

-Adjustment of the titer of virus solution-
The obtained virus solution was measured for infectious titer by X-gal staining using the activity of the lacZ gene contained in the vector backbone, and a helper virus (AdNG163R-2) was added accordingly, and the strain was further reintroduced into 116 strains. Amplified by infection. At the stage of the fourth virus solution, it was recovered as a band by cesium chloride density gradient centrifugation and dialyzed. The virus concentrate was measured for infectious titer by X-gal staining and physical titer by genomic Southern hybridization (“Palmer DJ, Ng P. Physical and infectious titers of dependent-dependent vectors: a of direct comparison to the adenovirus reference material. Mol Ther 2004; 10 (4): 792-8.). As a result, a virus concentrate having a physical titer of about 1 × 10 ¹⁰ to 1 × 10 ¹¹ was obtained.

(Comparative Production Example 1: Helper-dependent adenovirus vector-2)
HDAdVenus-geo (β-galactosidase and neomycin packaged in 116 cells using AdNG163 as a helper virus as a control vector that constitutively expresses the fluorescent protein Venus (hereinafter sometimes referred to as “HDAd-CAG-Venus”) fusion gene) -TK was used (Suzuki K of the resistance gene, Mitsui K, Aizawa E et al. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci U S a 2008; 1 5 (37):. 13781-6 reference).

-Adjustment of the titer of virus solution-
In the same manner as in Production Example 1, a virus concentrate having a physical titer of about 1 × 10 ¹⁰ to 1 × 10 ¹¹ was obtained.

(Comparative Production Example 2: E1 / E3-deficient adenovirus vector-1)
As an E1 / E3-deficient adenovirus vector-1 (about 35 kb), a promoter from the translation start point of the first exon of the human osteocalcin gene region to 3.8 kb of the 5 ′ upstream region (see SEQ ID NO: 37 (3, 3) A vector in which the Venus gene, which is a fluorescent protein gene, was linked to 722 bp)) was produced as follows.
The helper-dependent adenovirus vector plasmid (pAMHDAdGT-hOC-Venus) is digested with restriction enzymes HindIII and NotI, and subjected to agarose gel electrophoresis, 5 ′ upstream from the translation start point of the first exon of the human osteocalcin gene region A 4.7 kb fragment containing 3.8 kb of the region, Venus gene, and pA addition sequence was excised, and pShuttle (“He TC, Zhou S, da Costa LT et al. A simplified system” was used as a cloning plasmid vector. for generating recombinant adenoviruses. See Proc Natl Acad Sci USA 1998; 95 (5): 2509-14.)). It was inserted by ligation reaction into the NotI restriction enzyme site. The shuttle vector plasmid thus obtained (hereinafter sometimes referred to as “pShuttle-hOC3.8-Venus”, 11.2 kb) is digested with the restriction enzyme PmeI to form a linear form, and E1 / E3-deficient adenovirus is obtained. By introducing it into BJ5183-AD-1 (obtained from Agilent Technologies), which is an Escherichia coli strain carrying the vector plasmid pAdEasy-1 (see the above-mentioned pShuttle literature, obtained from Agilent Technologies), By causing homologous recombination between the homologous sequences, an E1 / E3-deficient adenovirus vector plasmid (hereinafter referred to as “pAdEasyShuttle-hOC3.8”) that retains the 3.8 kb of the 5 ′ upstream region of the human osteocalcin gene and the Venus gene. -Venus " May be called). The E1 / E3-deficient adenovirus vector plasmid was linearized by digestion with the restriction enzyme PacI, and transiently introduced into 293A cells by lipofection, and packaged as an adenovirus.
FIG. 6 shows a schematic diagram of E1 / E3-deficient adenovirus vector-1. In FIG. 6, “Ad5 (ΔE1 / 3” indicates the genome sequence of type 5 adenovirus in which the E1 gene and E3 gene are deleted, “Venus” indicates the fluorescent protein Venus gene, and “Ψ” indicates the package. The “double-ended arrow” indicates the terminal inverted sequence (ITR) of the adenoviral vector.

-Adjustment of the titer of virus solution-
The infectious titer of the virus solution was evaluated by the cytopathic effect (CPE (Cytopathic Effect)) observed at the time of infection of 293A cells, and a minimum amount of virus solution that denatures 293A cells almost 100% was applied to 293A cells. While infecting, the virus was amplified and purified in the same manner as in Preparation Example 1 to obtain a virus concentrate having a physical titer of about 1 × 10 ¹¹ to 1 × 10 ¹² .

(Comparative Production Example 3: E1 / E3-deficient adenovirus vector-2)
A control vector that constitutively expresses the fluorescent protein GFP was produced as follows.
As a control shuttle vector plasmid for the AdEasy method used in Comparative Production Example 2, pAdTrack (see pShuttle literature described in Comparative Production Example 2) that expresses the fluorescent protein gene EGFP by the CMV promoter was obtained by the same method. The control E1 / E3-deficient adenovirus vector plasmid (hereinafter referred to as pAdEasyTrack) constructed and incorporated in No. 1 was linearized and introduced into 293A cells in the same manner as the adenovirus vector of Comparative Production Example 2, and as an adenovirus. Packaged.

-Adjustment of the titer of virus solution-
In the same manner as in Comparative Production Example 2, a virus concentrate having a physical titer of about 1 × 10 ¹¹ to 1 × 10 ¹² was obtained.

(Test method)
In each of the following test examples, the test was performed as follows.

<Preparation of primary osteoblasts and formation of bone-like nodules>
-Preparation of primary osteoblasts-
Mouse primary osteoblasts (hereinafter sometimes referred to as "POB") is, "Ohyama Y, Nifuji A, Maeda Y et al. Spaciotemporal association and bone morphogenetic protein regulation of sclerostin and osterix expression during embryonic osteogenesis. Endocrinology 2004 145 (10): 4685-92. "And" Nefusi JR, Brami G, Modrowski D et al. Sequential expression of bone matrix protection calibration rat calves. "Differentiation and bone formation in vitro. J Histochem Cytochem 1997; 45 (4): 493-503."

The top of the skull was taken out from a newborn of the mouse inbred line C57BL / 6J Jc1 (manufactured by Claire Japan). The top of the head is a 1 × phosphate buffer solution (hereinafter referred to as “PBS”) containing 0.1% collagenase (Wako Pure Chemical Industries, Ltd.) and 0.2% dispase (God Shusei Co., Ltd.) After the digestion at 37 ° C. for 10 minutes, the washing with 1 × PBS was repeated 5 times.
Most of the first wash was fibroblasts and was discarded, and the second and subsequent washes were used as primary osteoblasts.

-Formation of bone-like nodules-
The primary osteoblasts are suspended in 10% FCS-containing αMEM (Life Technologies) containing 50 U / mL streptomycin (Life Technologies) and 50 μg / mL penicillin (Life Technologies), and coated with gelatin. The 6-well plastic dish (manufactured by Griener) is seeded at a density of 2.0 × 10 ⁶ cells / well, and the 8-well slide chamber (manufactured by Iwaki) is seeded at a density of 3.2 × 10 ⁵ cells / well. The cells were cultured at 37 ° C. with 5% CO ₂ .
The medium was changed every 48 hours for 1 to 2 weeks until bone-like nodules were formed.

<Virus infection of MG-63 cells or primary osteoblasts>
-Virus infection of MG-63 cells-
Virus infection of MG-63 cells, "Suzuki K, Mitsui K, Aizawa E et al. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci U S A 2008 105 (37): 13781-6. ”And carried out as follows.

The day before infection, MG-63 cells were seeded in 12-well plates.
On the day of infection, the medium was replaced with 200 μL of 1 × PBS containing Mg ²⁺ and Ca ²⁺ (hereinafter sometimes referred to as “PBS (++)”). Hereinafter, it may be referred to as “MOI”) was diluted with PBS (++) so as to be 10,000.
After incubation for 1 hour at room temperature, medium containing 10% FCS was added.
In the helper-dependent adenovirus vector-2 obtained in Comparative Production Example 1, 99% of the cells were transduced with the MOI.

--- 1α, 25 (OH) ₂ D ₃ administration-
In a test for examining the effect of the osteocalcin promoter, 1α, 25 (OH) ₂ D ₃ (manufactured by Sigma-Aldrich, hereinafter sometimes referred to as “VD3”) is added to a culture containing cells infected with an adenovirus vector. The final concentration was 0 nM, 1 nM, 10 nM, or 100 nM, and the cells were cultured.

-Virus infection of primary osteoblasts-
Virus infection of the primary osteoblasts is, "Suzuki K, Mitsui K, Aizawa E et al. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors. Proc Natl Acad Sci U S A 2008; 105 (37): 13781-6. ”And carried out as follows.

Excluding the primary osteoblast culture medium in which bone-like nodules were formed by the above-mentioned “formation of bone-like nodules”, PBS (++) of ¼ volume of the original medium and adenoviral vector were used in physical titer. In addition to an MOI of 1,000, infection was carried out with gentle shaking for 1 hour at room temperature.
One hour later, 2.0 mL of a 6-well plastic dish and 250 μL of a medium in an 8-well slide chamber were added and cultured overnight at 37% at 5% CO ₂ and then the medium was changed.
In addition, when the helper-dependent adenovirus vector-2 having a constant expression promoter produced in Comparative Production Example 1 was used at an MOI of 1,000, the expression of the fluorescent protein was confirmed under a fluorescence microscope 2 days after infection. It was done. In the helper-dependent adenovirus vector-2 produced in Comparative Production Example 1, 60% of cells were transduced with the MOI.

<Fluorescent immune antibody staining>
In order to compare the expression of the fluorescent protein by the adenovirus vector and the expression of endogenous osteocalcin, fluorescent immunoantibody staining was performed as follows.
Primary osteoblasts that formed bone-like nodules infected with virus in the 8-well slide chamber were removed on the third day after virus infection, washed twice with 1 × PBS, and fixed with 10% formalin solution for 10 minutes. It was done.
Next, after further washing twice with 1 × PBS, blocking was performed with a blocking solution (PBS containing 10% goat serum and 0.3% Triton-X 100) for 1 hour, and then diluted 1: 100 as a primary antibody. The anti-osteocalcin antibody (LB-4005 manufactured by LSL, rabbit, polyclonal) was reacted for 2 hours at room temperature.
Thereafter, the plate was washed twice with PBST, and reacted with Alexa Fluor 594-labeled anti-rabbit IgG antibody (manufactured by Life Technologies) diluted 1: 200 as a secondary antibody at room temperature for 2 hours.
Then, after washing twice with PBST, it was sealed with a mounting medium containing DAPI (4 ′, 6-diamidino-2-phenylindole) (ProLong Gold anti-reagent, manufactured by Life Technologies), and with a fluorescence microscope IX81 (manufactured by Olympus). Observed.

<FACS analysis and cell sorting>
FACS analysis and cell sorting were performed to sort cells expressing fluorescent protein (hereinafter sometimes referred to as “fluorescent protein positive cells”).
Primary osteoblasts or cells infected with the adenovirus vector were cultured for 2 days. On the 3rd day after virus infection, the plate was washed twice with PBS, peeled off with 0.05% Tripsin-EDTA (manufactured by Nacalai Tesque), suspended in PBS, centrifuged, and FACS buffer (2% FBS and 2 mM EDTA was added). In PBS).
FACS analysis was performed using a FACSCalibur flow cytometer (BD Biosciences).
Cell sorting was performed using a FACSAria II flow cytometer (BD Biosciences). Before sorting the cells, the cells were stained with propidium iodide (PI) to exclude dead cells. The sorted cells were precipitated by centrifugation, resuspended in FACS buffer, and checked for purity by performing a second flow cytometer. An uninfected culture was used for setting the fluorescence background level.

<Quantitative RT-PCR analysis>
RNA extraction for quantitative RT-PCR and cDNA synthesis are described in “Kokabu S, Nojima J, Kanomata K et al. Protein phosphatase phenide defensent in BMP”. Miner Res 2010; 25 (3): 653-60. "And" Suzuki K, Mitsui K, Aizawa E et al. Highly effective transgenic expression and generation of energy. . Th helper-dependent adenoviral vectors Proc Natl Acad Sci USA 2008; 105 (37):. 13781-6 "was carried out with reference to.

Total RNA was prepared by using DNase Mini Kit (Qiagen) and DNase treatment on the column.
In the reverse transcription reaction of the total RNA, PrimeScript 1st strand cDNA Synthesis Kit (manufactured by Takara Bio Inc.) was used to synthesize cDNA.

The RT-PCR reaction was analyzed using ABI PRISM 7000 (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems). In addition, the said analysis was each performed 3 times (n = 3).
A calibration curve was prepared using a sample obtained by serially diluting the cDNA, and a relative quantitative value was determined by a relative quantitative method.

Genes such as osteoblast markers (hereinafter sometimes referred to as “targets”) subjected to the RT-PCR reaction, and primer SEQ ID NOs are as follows. The sequence of each primer is also shown in Table 2.
(1) Target: Venus cDNA
Primer: SEQ ID NO: 5 (sometimes referred to as “Venus_GFP F”)
SEQ ID NO: 6 (sometimes referred to as “Venus_GFP R”)
(2) Target: human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA
Primer: SEQ ID NO: 7 (sometimes referred to as “RT-GAPDH-Fw2”)
SEQ ID NO: 8 (sometimes referred to as “RT-GAPDH-Rv2”)
(3) Target: human osteocalcin cDNA
Primer: SEQ ID NO: 9 (sometimes referred to as “hBGLP-f2”)
SEQ ID NO: 10 (sometimes referred to as “hBGLP-r2”)
(4) Target: mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA
Primer: SEQ ID NO: 11 (sometimes referred to as “Gapdh F”)
SEQ ID NO: 12 (sometimes referred to as “Gapdh R”)
(5) Target: mouse osteocalcin cDNA
Primer: SEQ ID NO: 13 (sometimes referred to as “mOG1-f2”)
SEQ ID NO: 14 (sometimes referred to as “mOG1-r2”)
(6) Target: Mouse Osx cDNA
Primer: SEQ ID NO: 15 (sometimes referred to as “Osx F”)
SEQ ID NO: 16 (sometimes referred to as “Osx R”)
(7) Target: mouse type I collagen a1 chain (ColIa1) cDNA
Primer: SEQ ID NO: 17 (sometimes referred to as “ColIa1 F”)
SEQ ID NO: 18 (sometimes referred to as “ColIa1 R”)
(8) Target: mouse type I collagen a2 chain (ColIa2) cDNA
Primer: SEQ ID NO: 19 (sometimes referred to as “ColIa2 F”)
SEQ ID NO: 20 (sometimes referred to as “ColIa2 R”)
(9) Target: Mouse Runx2 cDNA
Primer: SEQ ID NO: 21 (sometimes referred to as “Runx2 F”)
SEQ ID NO: 22 (sometimes referred to as “Runx2 R”)
(10) Target: mouse alkaline phosphatase (ALP) cDNA
Primer: SEQ ID NO: 23 (sometimes referred to as “Alp F”)
SEQ ID NO: 24 (sometimes referred to as “Alp R”)
(11) Target: mouse parathyrido homone receptor (Pthlr) cDNA
Primer: SEQ ID NO: 25 (sometimes referred to as “Pth1r F”)
SEQ ID NO: 26 (sometimes referred to as “Pth1r R”)
(12) Target: mouse bone sialoprotein (BSP) cDNA
Primer: SEQ ID NO: 27 (sometimes referred to as “Bsp F”)
SEQ ID NO: 28 (sometimes referred to as “Bsp R”)
(13) Target: mouse secreted protein acid and rich in cysteine (SPARC; also called osteonectin) cDNA
Primer: SEQ ID NO: 29 (sometimes referred to as “Sarc F”)
SEQ ID NO: 30 (sometimes referred to as “Spark R”)
(14) Target: mouse osteoprotegerin (OPG) cDNA
Primer: SEQ ID NO: 31 (sometimes referred to as “Opg F”)
SEQ ID NO: 32 (sometimes referred to as “Opg R”)
(15) Target: mouse RANKL cDNA
Primer: SEQ ID NO: 33 (sometimes referred to as “Rank F”)
SEQ ID NO: 34 (sometimes referred to as “Rankl R”)

Among the primers described above, those for which the target is Venus cDNA, human osteocalcin cDNA, human GAPDH cDNA, mouse Osx cDNA, mouse osteocalcin cDNA are primer designing software, Oligo 7 (Molecular Biology Insights, Ca.). Designed.
Among the primers, the target is mouse GAPDH cDNA, mouse ColIa1 cDNA, mouse ColIa2 cDNA, mouse Runx2 cDNA, mouse ALP cDNA, mouse SPARC cDNA, mouse Pthlr cDNA, mouse BSP cDNA, mouse OPG cDNA, mouse RANKL cDNA Was designed using a Perfect Real Time support system (manufactured by Takara Bio Inc.).

(Test Example 1)
Using the human osteosarcoma cell line MG-63 cells and the helper-dependent adenovirus vector-1 obtained in Production Example 1, the expression of the fluorescent protein Venus gene introduced into the human osteocalcin gene region was examined. As a negative control, cells that were not infected with virus were used.

<Fluorescence microscope observation>
FIG. 2A shows the result of observing the cells on the second day after the addition of VD3 with a fluorescence microscope (Olympus, 20 × objective lens, exposure time 1 second).
In FIG. 2A, “No infection” indicates the result of the negative control in which virus infection was not performed, and “hOC-Venus” indicates the cell infected with the helper-dependent adenovirus vector-1 obtained in Production Example 1. The scale bar in the right figure shows 100 μm.
From the results of FIG. 2A, Venus positive cells were observed in the MG-63 cells infected with the helper-dependent adenoviral vector-1 obtained in Production Example 1, and the average per VD3 dose-dependently. The amount of fluorescence increased.

<FACS analysis>
The result of FACS analysis is shown in FIG. 2B. The vertical axis of FIG. 2B shows the average fluorescence intensity of all cells. In FIG. 2B, “No infection” indicates the result of the negative control in which no virus infection was performed, and “hOC-Venus” indicates the cells infected with the helper-dependent adenovirus vector-1 obtained in Production Example 1. The results are shown.

<Quantitative RT-PCR analysis>
Fluorescent protein positive cells were separated by FACS, and quantitative RT-PCR analysis was performed on the fluorescent protein Venus gene and endogenous osteocalcin gene introduced into the human osteocalcin gene region using the sorted cells.
FIG. 2C shows the results of quantitative RT-PCR analysis of the fluorescent protein Venus gene introduced into the human osteocalcin gene region, and FIG. 2D shows the results of quantitative RT-PCR analysis of the endogenous osteocalcin gene. . In FIGS. 2C and 2D, the vertical axis indicates the relative mRNA level after normalization with GAPDH as an internal standard. 2C and 2D, “No infection” shows the result of negative control in which no virus infection was performed, and “hOC-Venus” was infected with the helper-dependent adenovirus vector-1 obtained in Production Example 1. The result of the made cell is shown.

From the above results, it was found that the average fluorescence intensity of all cells increased in a dose-dependent manner with VD3 in parallel with the endogenous osteocalcin mRNA level in MG-63 cells by VD3 treatment.

(Test Example 2)
The expression of the fluorescent protein Venus gene introduced into the human osteocalcin gene region is specific to mature osteoblasts. In primary osteoblasts forming bone-like nodules, the above-mentioned production example 1 or comparative production example 1 The obtained helper-dependent adenovirus vector was infected and examined. As a negative control, cells not infected with the virus were similarly examined. The results are shown in FIGS. 3A and 3B.

In FIG. 3A, “A hOC-Venus” indicates the result of infection with the helper-dependent adenovirus vector-1 obtained in Production Example 1, and “B CAG-Venus” is the result of Comparative Production Example 1. The result of infection with the obtained helper-dependent adenovirus vector-2 is shown, and “C No Infection” shows the result of negative control. In FIG. 3A, “Ph” is a result of imaging with a phase contrast microscope (20 × objective lens, exposure time 100 ms), “Venus” is a fluorescent image of the fluorescent protein Venus, and “Anti-OC”. Is a fluorescence image of an anti-osteocalcin antibody labeled with Alexa Fluor 594, and “DAPI” is a fluorescence image of what was encapsulated with a DAPI-containing encapsulant. Each fluorescence image is a result of photographing with a fluorescence microscope (20 × objective lens, exposure time 1 s). “Merge” is the result of merging “Venus”, “Anti-OC”, and “DAPI”. In addition, the scale bar in the lower right figure shows 50 micrometers.
FIG. 3B is a diagram showing a result obtained by imaging in the same manner as in FIG. 3A except that the image is taken using a 10 × objective lens and the scale bar in the lower right diagram is 100 μm. Terms and the like in FIG. 3B are the same as those in FIG. 3A.

As shown in FIG. 3A, when the helper-dependent adenovirus vector-1 obtained in Production Example 1 was infected, the fluorescent protein Venus was specifically expressed in cells forming bone-like nodules. . In addition, when the cells forming these bone-like nodules were immunostained with an anti-mouse osteocalcin antibody, the expression of the fluorescent protein Venus correlated with endogenous osteocalcin.
On the other hand, in the cells infected with the helper-dependent adenovirus vector-2 obtained in Comparative Production Example 1, the fluorescent protein Venus is expressed not only in cells forming bone-like nodules but also in surrounding cells. Was.

(Test Example 3)
Analysis of osteoblasts forming bone-like nodules in the culture was performed as follows.

<FACS analysis>
According to the FACS, osteoblasts may be referred to as a fluorescent protein positive cell group (hereinafter sometimes referred to as “Venus (+)”) and a fluorescent protein negative cell group (hereinafter referred to as “Venus (−)”). ) And separated. Cell sorting was performed by two-color FACS analysis of the fluorescent protein Venus and the autofluorescence (PE-A) of the cells.
The results when the helper-dependent adenovirus vector-1 obtained in Production Example 1 is used are shown in FIG. 4A. From the result of FIG. 4A, Venus (+) was 5.0% of the whole cell, and purity was 86.8%.
On the other hand, when the helper-dependent adenovirus vector-2 obtained in Comparative Production Example 1 was used, Venus (+) was about 60% (FIG. 4B), and when virus infection was not performed, Venus ( +) Was 0% (FIG. 4C).

<Quantitative RT-PCR analysis>
The mRNA expression level of genes related to bone formation and resorption was analyzed by quantitative RT-PCR analysis.
For the analysis, cells infected with the helper-dependent adenovirus vector of Production Example 1 (1) a group of cells that have not been sorted by the FACS (hereinafter referred to as “U group”) ), (2) a cell group that does not express the fluorescent protein Venus fractionated by the FACS (hereinafter sometimes referred to as “-group”), and (3) the fluorescent protein Venus fractionated by the FACS. Expressing cells (hereinafter sometimes referred to as “+ group”) were used. The results are shown in FIGS. 4D and 4E.

4D and 4E, “Gapdh” indicates GAPDH that is an internal standard, and “Venus” indicates the fluorescent protein Venus that is a reporter. “Oc” (osteocalcin), “Bsp” (BSP), “ColIa1”, “ColIa2”, and “SPARC” (SPARC) are secreted proteins, “Pth1r” is a hormone, “Alp” (ALP) Are enzymes, “Osx” and “Runx2” are transcription factors, and “Opg” (OPG) and “Rankl” (RANKL) are associated with the induction of osteoclasts.
The numerical values in the graphs of FIGS. 4D and 4E indicate the relative mRNA amount compared to the “− group”, and those without the numerical value indicate that the relative mRNA amount compared to the “− group” is approximately 1. 0.

From the results of FIGS. 4D and 4E, the expression level of the fluorescent protein Venus mRNA is 49.0 times higher in the “+ group” than in the “− group”, and mature osteoblasts can be sorted by FACS. Indicated. Further, in the “+ group”, not only the expression level of osteocalcin mRNA was 13.8 times higher but also the expression level of BSP mRNA was 19.3 times higher than that in the “− group”. However, the expression levels of ColIa1, ColIa2, and SPARC mRNA were almost the same between the “+ group” and the “− group”. The expression levels of Pth1r and ALP mRNA were 2.6 times and 2.2 times higher in the “+ group” than in the “− group”, respectively. The expression levels of OPG and RANKL mRNA were almost the same between the “+ group” and the “− group”.

The transcription factor Runx2, which was identified as an important transcription factor for osteoblast-specific expression of osteocalcin mRNA, was expressed at the mRNA level between the “+ group” and the “− group”. The difference was not seen.
On the other hand, the expression level of mRNA of Osx, which is another transcription factor, was 3.5 times higher in the “+ group” than in the “− group”.

From the above results, in the “+ group”, the differentiation markers specific to osteoblasts such as osteocalcin and BSP increased by 10 times or more compared to the “− group”. Expression of ALP, PTH receptor (Pth1r), etc., which rises as cells mature, is about 2 to 3 times higher, and the helper-dependent adenovirus vector-1 obtained in Production Example 1 is transiently osteoblastic. It was shown that the osteoblasts in the osteogenesis stage can be efficiently sorted from the cell populations in various differentiated states by virtue of the fluorescence intensity of the fluorescent protein Venus.

The Osx has been identified as a transcription factor necessary for osteogenic differentiation induced by BMP of C2C12 myoblasts ("Yagi K, Tsuji K, Nifuji A et al. Bone morphology"). protein-2 enhance ossterix gene expression in chondrocycles. J Cell Biochem 2003; 88 (6): 1077-83.)), and Osx knockout mice are known to lack bone tissue due to lack of osteoblast differentiation. ("Nakashima K, Zhou X, Kunkel G et al. The novel zinc finger-containing transcription factor osteo" .. Ix is required for osteoblast differentiation and bone formation Cell 2002; 108 (1): 17-29 "reference).
On the other hand, since Runx2 was expressed in progenitor cells of Osx knockout mice, it was suggested that Osx and Runx2 have different roles in osteoblast differentiation. The expression of Osx, ALP, PTH receptor, etc. is higher in mature osteoblasts than in immature osteoblasts. It can be said that this is a novel finding.

(Test Example 4)
<Fluorescent immune antibody staining>
The primary osteoblasts are infected with the helper-dependent adenovirus vector-1 obtained in Production Example 1 or the E1 / E3-deficient adenovirus vector-1 obtained in Comparative Production Example 2, and a fluorescent immune antibody Comparison was made by staining. As a control, fluorescent immunoantibody staining was similarly performed using the E1 / E3-deficient adenovirus vector-2 obtained in Comparative Production Example 3. The results are shown in FIG. 5A.

In FIG. 5A, “HDAd-hOC-Venus” shows the result of infection with the helper-dependent adenovirus vector-1 obtained in Production Example 1, and “ΔE1-hOC3.8-Venus” The result of infection with the E1 / E3-deficient adenovirus vector-1 obtained in Production Example 2 is shown, and “ΔE1-CMV-GFP” is the E1 / E3-deficient adenovirus obtained in Comparative Production Example 3 The results of infection with vector-2 (control) are shown. In FIG. 5A, “Phase” is a result of imaging with a phase contrast microscope (20 × objective lens, exposure time 100 ms), “Venus” is a fluorescent image of the fluorescent protein Venus, and “Anti-OC”. Is a fluorescence image of an anti-osteocalcin antibody labeled with Alexa Fluor 594, and “DAPI” is a fluorescence image of what is encapsulated with a DAPI-containing encapsulant. Each fluorescence image is a result of photographing with a fluorescence microscope (20 × objective lens, exposure time 1 s). “Merge” is the result of merging “Venus”, “Anti-OC”, and “DAPI”. The scale bar in the lower right figure indicates 100 μm.

From the results of FIG. 5A, both of “HDAd-hOC-Venus” and “ΔE1-hOC3.8-Venus” showed the expression of fluorescent protein Venus in bone-like nodules positive for anti-osteocalcin antibody. “HDAd-hOC-Venus” exhibited brighter fluorescence than “ΔE1-hOC3.8-Venus”. On the other hand, “ΔE1-CMV-GFP” used as a control showed high fluorescence in many cells other than bone-like nodules.

<FACS analysis>
The primary osteoblasts were infected with the helper-dependent adenovirus vector-1 obtained in Production Example 1 or the E1 / E3-deficient adenovirus vector-1 obtained in Comparative Production Example 2, and FACS analysis was performed. A comparison was made. As a control, when the E1 / E3-deficient adenovirus vector-2 obtained in Comparative Production Example 3 was used, the same test was conducted when no virus infection was performed. The results are shown in FIGS. 5B to 5E.

From the results of FIGS. 5B to 5E, when the helper-dependent adenovirus vector-1 obtained in Production Example 1 was used, the E1 / E3-deficient adenovirus vector-1 obtained in Comparative Production Example 2 was used. The amount of shining cells was 6 times higher than when used, and the average fluorescence intensity of the shining cells was also 3 times higher.

From these results, the helper-dependent adenovirus vector-1 obtained in Production Example 1 of the present invention is a previously reported paper ("Bilic-Curic I, Kronenberg M, Jiang X et al. Visualizing levels of osteoblast differentiation." two-color promoter-GFP strategy: Type I collagen-GFPcyan and osteocalcin-GFPtpz. Genesis 2005; 43 (2): 87-98., “Born AK, -LischerWursierK. . Ing of osteogenic differentiation using an osteocalcin reporter J Cell Biochem 2012; 113 (1):.. 313-21, "" Born AK, Rottmar M, Lischer S et al Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring (Eur Cell Mater 2009; 18: 49-60, 1-2; discussion]) with a Venus gene linked to a short 3.8 kb promoter (Comparative Production Example 2) Obtained are found to be superior to E1 / E3-deficient adenoviral vector -1). Further, from this result, the 5 'upstream region of the first exon of the osteocalcin gene, which is not included in the 3.8 kb promoter and is retained by the helper-dependent adenovirus vector-1 obtained in Production Example 1 above. There is an unknown expression control region involved in expression in mature osteoblasts in the sequence between 3.8 kb and 10.1 kb, or in the 8.8 kb sequence in the 3 ′ downstream region of the first exon of the osteocalcin gene The possibility was suggested.

From the results of each of the above test examples, the translation start point of the first exon of the osteocalcin gene including the human osteocalcin gene region of about 19 kb including not only the Runx2 and Vitamin D receptor binding sequences but also the upstream region and the downstream region. By using the helper-dependent adenovirus vector according to one embodiment of the present invention in which the fluorescent protein Venus gene is inserted into a cell population in various differentiated states by being transiently infected with this virus, It has been shown that only the osteoblasts present in can be sorted.

In addition, gene transfer via the adenovirus of the present invention is more effective in various cells than other viral vectors or non-viral methods.
So far, transgenic mice expressing GFP under the control of the 3.8 kb human osteocalcin promoter have been shown to express GFP specifically in mature osteoblasts ("Bilic-Curic I, Kronenberg M"). , Jiang X et al., Visualizing levels of osteoblast differentiation by a two-color promoter-GFP strategy: is-200, in-the-G.p.
On the other hand, the helper-dependent adenoviral vector of the present invention is useful in that it can be used to visualize osteocalcin-dependent GFP expression in not only mouse cells but also species of cells including humans.

In addition, there is an example in which a construct in which GFP is located under a 3.8 kb osteocalcin promoter is transiently introduced into human bone marrow-derived cells using nucleofection, but the introduction efficiency is at most 25%. ("Born AK, Lischer S, Maniura-Weber K. Watching osteogenesis: life monitoring of osteogenetic reporter. , Lischer Set et al. Correlating cell architecture with osteogenesi s: first steps lives live single cell monitoring. See Eur Cell Mater 2009; 18: 49-60, 1-2;
On the other hand, as shown in the above test example, the infection efficiency of the adenovirus vector is 60% when the MOI is 1,000, which is higher than that of the nucleofection.

The helper-dependent adenovirus vector of the present invention utilizes the large cloning capacity of helper-dependent adenovirus. That is, in one embodiment of the helper-dependent adenovirus vector of the present invention, the expression of the fluorescent protein gene is 10.1 kb in the 5 ′ upstream region of the first exon of the human osteocalcin gene and 8. in the 3 ′ downstream region. It is controlled by a long control sequence of 8 kb.
To date, there is an example of a GFP transgenic mouse using a short control sequence called a 1.7 kb rat osteocalcin promoter. In this example, the GFP signal is weak even in mature osteoblasts, and leakage expression is expressed in the central nervous system. which was ( "Kalajzic Z, Liu P, Kalajzic I et al Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts:.. comparison between rat type I collagen and rat osteocalcin promoters Bone 2002; 31 (6): 654-60 ."reference).
On the other hand, when the helper-dependent adenovirus vector of the present invention is used, the expression of endogenous osteocalcin can be more accurately reflected as shown in the test examples described above.
In addition, helper-dependent adenoviral vectors have sufficient capacity to accept long regulatory elements and have minimal viral enhancers that interfere with reporter gene expression, so that first generation adenoviruses are expressed in tissue-specific expression. It is better than viral vectors ( "Shi CX, Hitt M, Ng P et al. superior tissue-specific expression from tyrosinase and prostate-specific antigen promoters / enhancers in helper-dependent compared with first-generation adenoviral vectors. Hum Gene Ther 2002; 13 (2): 211- 4. "reference).

Furthermore, the helper-dependent adenovirus vector of the present invention enables transient expression of the fluorescent protein without incorporating the fluorescent protein gene into the chromosome of the target cell. Therefore, unlike the case of using a retrovirus or a lentivirus, the DNA of the helper-dependent adenovirus vector is finally lost from the infected cells by cell division. This feature is a great advantage in clinical application of the helper-dependent adenoviral vector of the present invention (for example, sorting of mature osteoblasts for transplantation).

Examples of the aspect of the present invention include the following aspects.
<1> The base sequence of the first exon of the osteocalcin gene is replaced with a base sequence containing a reporter gene,
At least part of the 5 ′ upstream region of the first exon of the osteocalcin gene;
A helper-dependent adenovirus vector comprising at least part of the 3 ′ downstream region of the first exon of the osteocalcin gene.
<2> The base sequence containing the reporter gene is located between at least a part of the 5 ′ upstream region of the first exon of the osteocalcin gene and at least a part of the 3 ′ downstream region of the first exon of the osteocalcin gene. The helper-dependent adenovirus vector according to <1>.
<3> The helper dependence according to any one of <1> to <2>, wherein the 5 ′ upstream region of the first exon of the osteocalcin gene is a region from the translation start point of the first exon to 10.1 kb. Type adenovirus vector.
<4> The helper-dependent adeno according to any one of <1> to <3>, wherein the 3 ′ downstream region of the first exon of the osteocalcin gene is a region from the end of the first exon to 8.8 kb. Viral vector.
<5> The helper-dependent adenovirus vector according to any one of <1> to <4>, wherein the base sequence including the reporter gene includes a base sequence of an antibiotic resistance gene.
<6> a step of infecting osteoblasts with the helper-dependent adenovirus vector according to any one of <1> to <5>;
A method for visualizing mature osteoblasts, comprising the step of detecting the expression of a reporter gene in the infected osteoblasts.
<7> Infecting osteoblasts with the helper-dependent adenovirus vector according to any one of <1> to <5>,
Detecting the expression of a reporter gene in the infected osteoblasts;
A method for producing mature osteoblasts comprising the step of sorting cells in which expression of the reporter gene is detected.

Since the helper-dependent adenovirus vector of the present invention can visualize mature osteoblasts in a bone-forming state, it can be suitably used for the method for visualizing and producing mature osteoblasts. The helper-dependent adenoviral vectors of the present invention not only examine mature osteoblasts in vitro, but also manipulate mature osteoblasts in vivo (eg, cell-based techniques for bone regeneration). It is considered useful.
Since the method for visualizing mature osteoblasts of the present invention can visualize mature osteoblasts in a bone-forming state, it can be suitably used for searching for new bone formation control factors.
The method for producing mature osteoblasts of the present invention can sort mature osteoblasts in a bone-forming state, which has been difficult in the past, in a living state, and therefore can be applied to tissue regeneration using osteoblasts. Can be expected.

Claims (7)

1. The base sequence of the first exon of the osteocalcin gene is replaced with a base sequence containing a reporter gene,
   At least part of the 5 ′ upstream region of the first exon of the osteocalcin gene;
   A helper-dependent adenovirus vector comprising at least a part of the 3 ′ downstream region of the first exon of the osteocalcin gene.
2. The base sequence containing the reporter gene is located between at least a part of the 5 ′ upstream region of the first exon of the osteocalcin gene and at least a part of the 3 ′ downstream region of the first exon of the osteocalcin gene. 2. The helper-dependent adenovirus vector according to 1.
3. The helper-dependent adenovirus vector according to any one of claims 1 to 2, wherein the 5 'upstream region of the first exon of the osteocalcin gene is a region from the translation start point of the first exon to 10.1 kb.
4. The helper-dependent adenovirus vector according to any one of claims 1 to 3, wherein the 3 'downstream region of the first exon of the osteocalcin gene is a region from the end of the first exon to 8.8 kb.
5. The helper-dependent adenovirus vector according to any one of claims 1 to 4, wherein the base sequence comprising the reporter gene comprises a base sequence of an antibiotic resistance gene.
6. Infecting osteoblasts with the helper-dependent adenoviral vector according to any one of claims 1 to 5;
   A method for visualizing mature osteoblasts, comprising the step of detecting expression of a reporter gene in the infected osteoblasts.
7. Infecting osteoblasts with the helper-dependent adenoviral vector according to any one of claims 1 to 5;
   Detecting the expression of a reporter gene in the infected osteoblasts;
   A method for producing mature osteoblasts comprising the step of sorting cells in which expression of the reporter gene is detected.

Images