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TECHNOLOGY DEVELOPMENT

Establishment of the Gene-Inducible System in Primate Embryonic Stem Cell Lines

^aDepartment of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;
^bLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;
^cDepartment of Gastroenterological Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan

Key Words. Primate embryonic stem cells • Gene-inducible system • Tetracycline • Tet-Off system • Doxycycline Enhanced green fluorescent protein

Correspondence: Hirofumi Suemori, Ph.D., Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shougoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: +81-75-751-3821; Fax: +81-75-751-3890; e-mail: hsuemori{at}frontier.kyoto-u.ac.jp

Received December 29, 2005; accepted for publication July 14, 2006.
First published online in STEM CELLS EXPRESS   July 20, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 
Human embryonic stem cells (ESCs) would provide a potentially unlimited source for cell replacement therapies. However, the molecular mechanisms involved in the maintenance of "stemness" are not fully understood. Monkey ESCs are much more similar in character to human ESCs than are mouse ESCs. Therefore, studies using monkey ESCs can give conclusions that are more relevant and may be readily applicable to both basic research and clinical applications for future regenerative medicine. For such studies, generation of a gene-inducible system regulatable in primate ESCs would serve as a powerful tool. Here, we established a Tet-Off gene-inducible system in monkey ESC lines. Such manipulated cells maintained ESC characteristics, and inducible gene expression in both the stem cells and differentiated cells could be reliably controlled by doxycycline administration.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 
Embryonic stem cells (ESCs) are capable of self-renewal and pluripotency. They can differentiate into cell types of all three embryonic germ layers: endoderm, mesoderm, and ectoderm. The first ESC lines were established from the inner cell mass of mouse blastocysts [1, 2]. Then, ESC lines from nonhuman primates, rhesus monkey (Macaca mulatta) [3], cynomolgus monkey (Macaca fascicularis) [4], and humans [5] were established. Due to the phylogenetical closeness between monkeys and humans, monkey ESCs serve as invaluable models for both basic science research and preclinical applications that, because of practical and ethical limitations, cannot be conducted in humans [6]. Thus, monkey ESCs provide research tools by which to understand properties and behaviors of human ESCs.

Although genetic engineering, including efficient gene delivery, may be essential for the application of ESCs, it has proven to be more difficult to manipulate primate ESCs than mouse ESCs. Constitutive overexpression of a transgene directed by strong promoters, such as the phosphoglycerate kinase-1 gene promoter or cytomegalovirus (CMV) immediate early gene promoter, has revealed the functions of numerous genes in ESCs [7]. More recently, knockdown of a gene using small-interfering RNAs has been useful for understanding the gene function [8, 9]. These methods, however, often fail to detect molecular mechanisms that are dependent on the precise levels of the gene expression. Inducible gene expression, which makes possible both the regulated overexpression and repression of a transgene, is a useful technique that needs to be established in primate ESCs. Such a technique may prove to be a valuable tool advancing the safety and efficacy of gene therapy. The most commonly used system in mammals is a tetracycline-inducible gene expression system (Tet-Off and Tet-On) [10, 11]. The Tet-Off and Tet-On systems offer many advantages, including strict temporal on/off regulation, cell type- or tissue-specific control of expression, and a well-characterized inducer, tetracycline or its analog doxycycline (Dox) [12–14]. For example, quantitative expression of Oct-3/4 controls differentiation or self-renewal in mouse ESCs, demonstrating the importance of strict gene regulation [15]. The ROSA-TET system that has a knock-in step and exchange step is an efficient system for establishing multiple mouse ESC lines carrying a tetracycline-regulatable transgene at the ROSA26 locus [16]. Establishment of an optimal inducible gene expression system using gene targeting techniques has been possible in mouse ESC lines because of the existence of the ROSA26 locus targeting site, which is highly resistant to gene silencing [17]. However, there have not been any reports that indicate the presence of such loci that can escape gene silencing in primate ESCs. In addition to difficulties introducing exogenous genes into primate ESCs, the absence of an efficient targeting site makes it difficult to establish reliable inducible gene expression systems in primate ESC lines.

In this study, we present the first report of a reliable Tet-Off gene-inducible system in primate ESC lines. By monitoring enhanced green fluorescent protein (EGFP) expression as an indicator, we demonstrated that EGFP expression can be clearly controlled by Dox treatment in ESCs, embryoid bodies (EBs), and teratomas.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 
Culture of Cynomolgus Monkey ESCs
The cynomolgus monkey ESC line CMK6 was established and cultured as previously described [4]. Briefly, ESCs were cultured as colonies on mouse embryonic fibroblast (MEF) feeder cells treated with mitomycin C (Wako Pure Chemicals, Osaka, Japan, http://www.wako-chem.co.jp) and subcultured every 3–5 days by enzymatic dissociation.

Construction of Expression Plasmids
To create the regulator plasmid pCAG-tTA^Off-hyg, we inserted a CAG promoter [18] and tetracycline-controlled transactivator (tTA) sequence from the pTet-Off vector (Clontech, Mountain View, CA, http://www.clontech.com) into the pTK-Hyg vector (Clontech). To create the response plasmid pTRE-EGFP-neo, we inserted an EGFP sequence as a gene of interest into the multiple cloning site of the P_tight sequence from the pTRE-Tight vector (Clontech) and additionally inserted a neomycin-resistant gene with an SV40 promoter.

Transfection
Transfection of cynomolgus monkey ESCs was performed as previously described [19]. Briefly, ESCs, plated on hygromycin-resistant SL10 (subline of STO) cell lines treated with 10 ng/ml basic fibroblast growth factor (human recombinant) (Upstate Biotechnology, Lake Placid, NY, http://www.upstatebiotech.com), were transfected with linearized pCAG-tTA^Off-hyg (10 µg/60-mm tissue culture dish) in Opti-MEM (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) using Lipofectamine 2000 (Invitrogen) and selected in the presence of 30–50 µg/ml Hygromycin B (Invitrogen). After the selection, hygromycin-resistant colonies were picked up and transferred to MEF feeder cells. The regulator-transfected ESCs plated on neomycin-resistant MEF feeder cells were further transfected with linearized pTRE-EGFP-neo in the absence of Dox (Wako Pure Chemicals) and selected in the presence of 100 µg/ml G418 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). After the selection, neomycin-resistant colonies were picked up and transferred to new MEF feeder cells.

To regulate expression of EGFP, 1 ng/ml Dox was used unless otherwise indicated. For Dox removal, the ESCs were washed with phosphate-buffered saline (PBS) twice and incubated for 3 hours without Dox in the medium, and then the medium was changed again.

Histochemical Analyses of Stem Cell Markers
To detect alkaline phosphatase (AP) activity, ESCs were fixed with 3.7% formaldehyde in PBS and detected using Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

To detect Oct-3/4 expression, cells were fixed with 4% paraformaldehyde in PBS. After being washed with PBS, cells were treated with 0.2% Triton X-100 (nacalai tesque, Kyoto, Japan, http://www.nacalai.co.jp) in PBS for 10 minutes to permeabilize and then with 0.3% hydrogen peroxide (Wako Pure Chemicals) in PBS for 10 minutes to inactivate endogenous peroxidases. Cells were incubated with mouse anti-Oct-3/4 monoclonal antibody (clone C-10, 1:300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) for 1 hour. After incubation with horseradish peroxidase-conjugated goat anti-mouse immunoglobulins polyclonal antibody (1:300; Dako Denmark A/S, Glostrup, Denmark, http://www.dako.com) for 1 hour, Oct-3/4 localization was visualized using 3,3'-diaminobenzidine (DAB) (Sigma-Aldrich) as a substrate. The staining patterns of AP and Oct-3/4 were examined by light microscopy.

Immunocytochemical and Immunohistochemical Analyses
Cells fixed with 4% paraformaldehyde in PBS were permeabilized with 0.2% Triton X-100 in PBS and then incubated with 2% bovine serum albumin (BSA) (Sigma-Aldrich) and 3% normal goat serum (Chemicon International, Temecula, CA, http://www.chemicon.com) in PBS for herpes simplex virus VP16, GFP and neural cell adhesion molecule (NCAM) staining or with 5% BSA in PBS for -fetoprotein (AFP) and Desmin staining. Cells were incubated with rabbit anti-VP16 polyclonal antibody (1:500–1,000; Clontech), mouse anti-VP16 monoclonal antibody (clone 1-21, 1:50; Santa Cruz Biotechnology, Inc.), mouse anti-AFP monoclonal antibody (clone C3, 1:50; Sigma-Aldrich), rabbit anti-Desmin polyclonal antibody (1:50; Sigma-Aldrich), and rabbit anti-NCAM polyclonal antibody (1:400; Chemicon International) in PBS containing 2% BSA for either 1 hour at room temperature or overnight at 4°C. After incubation with Alexa Fluor 488- or 546-conjugated goat anti-rabbit or anti-mouse immunoglobulin G polyclonal antibody (1:300; Invitrogen) in PBS containing 2% BSA for 30–40 minutes, nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and analyzed by fluorescence microscopy.

Flow Cytometry
After treatment with varying concentrations of Dox (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, and 1,000 ng/ml), ESCs were dissociated into single-cell suspensions using 0.25% trypsin-1 mM EDTA. After resuspension in 3% knockout serum replacement (Invitrogen) in PBS, cells were analyzed on a BD FACSVantage SE flow cytometer (BD Biosciences, Franklin Lakes, NJ, http://www.bdbiosciences.com).

Formation of EBs
ESCs were detached from the feeder cells by enzymatic dissociation with gentle pipetting to avoid the dissociation of colonies. The cells were then cultured in suspension in a 100-mm Petri dish. EBs were grown in ESC medium in the presence or absence of 1 ng/ml Dox for 12 or 30 days, respectively, and then collected for total RNA preparation.

To examine EGFP expression in EBs, 12-day-old EBs fixed in 2% paraformaldehyde in PBS for 3 hours on ice were embedded in OCT Compound (Sakura Finetek Japan, Tokyo, http://www.sakura-finetek.com), frozen, cryosectioned to 10 µm, and examined by fluorescence microscopy.

For further spontaneous differentiation, the 12-day-old EBs were dissociated with 0.05% trypsin-1 mM EDTA, plated on Human Fibronectin Cellware 4-Well CultureSlides (BD Biosciences) for VP16 and NCAM staining, or 0.1% gelatin (type A; Sigma-Aldrich)-coated six-well tissue culture plates for AFP and Desmin staining, and cultured in Dulbecco's modified Eagle's medium (D5796; Sigma-Aldrich) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, http://www.jrhbio.com) and 1 mM sodium pyruvate (Sigma-Aldrich). Immunostaining was performed as described above.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted from feeder cell-less ESCs, 12-day-old, or 30-day-old EBs using RNeasy kit (Qiagen, Valencia, CA, http://www.qiagen.com). cDNA was synthesized from 2 µg of total RNA using Omniscript Reverse Transcriptase (Qiagen). Polymerase chain reactions (PCR) were optimized to facilitate a semiquantitative comparison of samples within the log phase of amplification. Gene-specific primers were designed based on published sequences (supplemental online Table 1). PCR products were separated on 1.5% agarose gel and visualized by ethidium-bromide staining.

Formation of Teratomas
ESCs were injected subcutaneously into severe combined immunodeficient (SCID) mice (CLEA Japan, Tokyo, http://www.clea-japan.com). For Dox treatment, mice were given 500 µg/ml Dox dissolved in drinking water containing 3% sucrose, which was changed twice a week. All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Kyoto University. Sixty to 70 days after injection, formed teratomas were examined for EGFP expression by fluorescence stereomicroscopy. Then, the teratomas were fixed in 3.7% formaldehyde in PBS overnight, embedded in OCT compound, frozen, and cryosectioned to 10 µm. EGFP expression was examined by fluorescence microscopy, and tTA expression was detected by VP16 immunostaining as described above. For histological analysis, fixed teratomas were embedded in paraffin, sectioned to 5 µm, and stained with hematoxylin and eosin.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 
Construction of the Regulator and Response Plasmids
The tetracycline-inducible system can analyze the effect of regulated overexpression or repression of a transgene. Although there are two ways in which to use this technology (i.e., Tet-Off and Tet-On), we focused on the use of the Tet-Off system because the Tet-On system often exhibits leaky expression under noninducing conditions. In addition, a higher concentration of Dox is often required in the Tet-On system for the regulation of gene expression than in the Tet-Off system [20]. To establish a Tet-Off system in monkey ESC lines, we constructed the regulator plasmid pCAG-tTA^Off-hyg and response plasmid pTRE-EGFP-neo (Fig. 1A) as described in Materials and Methods. We used the CAG promoter to drive tTA because it is highly active in a wide range of cell types, including ESCs, in which gene silencing frequently occurs [18]. The Tet-Off system uses tTA, a fusion protein of the VP16 activation domain and Tet repressor. This tTA induces transcription of EGFP in the absence of Dox by binding to the modified Tet-Response Element (TRE_mod), which is upstream of an altered minimal immediate early CMV promoter (P_minCMV).

View larger version (51K): [in this window] [in a new window]   Figure 1. Generation of the gene-inducible system in monkey ESC lines. (A): The regulator and response plasmid constructions for the Tet-Off system. The pCAG-tTAOff-hyg regulator plasmid expresses tTA under the control of the CAG promoter and has the hygromycin-resistant gene. The pTRE-EGFP-neo response plasmid expresses EGFP under the control of the TRE and has the neomycin-resistant gene. The Ptight contains modified TRE (TREmod) upstream of the altered minimal cytomegalovirus promoter (PminCMV). Only after tTA binds to the TREmod in the absence of Dox, transcription of EGFP can be activated. (B): Characteristics of the clone transfected with the regulator plasmid (clone #J). The regulator-transfected ESC clone exhibited almost homogeneous tTA expression by VP16 (a part of tTA) immunostaining in the nuclei, and this was maintained after more than 40 passages, including a few cycles of freeze/thawing. Nuclei were counterstained with DAPI. This clone demonstrated ESC-specific morphologies with a high nucleus-to-cytoplasm ratio and prominent nucleoli (phase contrast, higher magnification). These cells maintained markers such as AP activity and Oct-3/4 expression. (C, D): Characteristics of the clone transfected with both the regulator and response plasmids (clone #J-d). The double-transfected ESC clone expressed EGFP homogeneously and retained almost homogeneous tTA expression, ESC-specific morphologies, AP activity, and Oct-3/4 expression in the absence of Dox (C). EGFP expression was almost undetectable within 5 days of treatment with 1 ng/ml Dox without changes in tTA expression, morphological characteristics, and expression of stem cell markers (D). Scale bars = 100 µm (B–D). Abbreviations: AD, activation domain; AP, alkaline phosphatase; DAPI, 4',6-diamidino-2-phenylindole; Dox, doxycycline; –Dox, without doxycycline in the medium; +Dox, with doxycycline in the medium; EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; hyg, hygromycin; neo, neomycin; tetR, tet repressor; TRE, tetracycline-response element; tTA, tetracycline-controlled transactivator; VP16, herpes simplex virus; pA, polyadenylation signal.

Next, a subset of these clones—including clone #J, which exhibited Dox responsiveness and almost homogeneous tTA expression—was transfected with the response plasmid to create double-transfected ESC clones. After G418 selection in the absence of Dox, clones were screened for EGFP expression by fluorescence microscopy. Of the 15 clones investigated, 12 clones (80.0%) displayed EGFP expression and Dox responsiveness. The double-transfected ESC clones retained ESC-specific morphologies and expression of markers such as AP activity and Oct-3/4. These results suggest that these clones were maintained in the undifferentiated state after double transfection. These clones displayed Dox-responsive EGFP expression and almost homogeneous tTA expression (Fig. 1C [–Dox], 1D [+Dox]). Because we obtained basically the same results from each clone, clone #J-d was used in the following experiments. This clone retained ESC characteristics and Dox-responsive EGFP expression for more than 30 passages in the presence of G418, including a few cycles of freeze/thawing (data not shown).

When approximately 3 x 10⁵ monkey ESCs per 60-mm dish were transfected with pCAG-tTA^Off-hyg, three to five stable transformants were usually obtained after selection with hygromycin. In the case of pTRE-EGFP-neo, 18–29 stable transformants were obtained after G418 selection.

Dox-Responsive EGFP Expression in the Double-Transfected ESC Clone, EBs, and Teratomas
To determine the minimum effective concentration of Dox, the EGFP-positive double-transfected ESC clone cultured in varying concentrations of Dox (0–1,000 ng/ml) in the medium was examined for EGFP expression by fluorescence microscopy and flow cytometry. After the addition of Dox, suppression of EGFP expression was observed in a dose-dependent manner. Cells treated with Dox concentrations of 0.001 or 0.01 ng/ml exhibited similar EGFP expression levels as those observed in untreated cells. Cells given 0.05 or 0.1 ng/ml exhibited lower levels of EGFP expression, whereas EGFP was almost undetectable in cells given 0.5, 1, or 1,000 ng/ml Dox (Fig. 2A). Therefore, we used 1 ng/ml Dox as an optimal concentration for in vitro experiments. The clone exhibiting Dox-responsive EGFP expression maintained almost homogeneous tTA expression, AP activity, and Oct-3/4 expression (Fig. 1C, 1D), indicating that Dox responsiveness did not affect ESC characteristics. EGFP downregulation in the cells at 1 ng/ml Dox was observed as early as 1 day after the treatment, becoming almost undetectable after 5 days (Fig. 2B). On the other hand, the EGFP expression became detectable within 1 day of Dox removal. After 5 days without Dox, EGFP expression increased significantly, at higher levels than those observed on the first day after Dox removal, but still at lower levels than those cultured continuously without Dox (Fig. 2C). Monkey ESCs are very sensitive to dissociation, and the cells require transfer as small clusters during subculture. In addition, primate ESCs usually need to be maintained in the presence of the feeder cells. Because Dox can bind to extracellular matrices and intracellular components [21], complete removal of Dox from primate ESC cultures is considered to be difficult.

View larger version (57K): [in this window] [in a new window]   Figure 2. Dox-responsive EGFP expression in monkey ESC lines with the Tet-Off system. (A): The double-transfected ESC clone exhibited a dose-dependent response to Dox after 5 days of treatment by flow cytometry. Cells treated with Dox concentrations of 0.001 or 0.01 ng/ml displayed levels of EGFP expression similar to those observed in untreated cells. Cells given 0.05 or 0.1 ng/ml exhibited lower levels of EGFP expression, whereas EGFP was almost undetectable in cells treated with 0.5, 1, or 1,000 ng/ml Dox. (B): EGFP downregulation was observed as early as 1 day after treatment with 1 ng/ml Dox, with additional downregulation after 2 days, becoming almost undetectable after 5 days. (C): Restoration of EGFP expression was detected within 1 day of Dox removal from the medium (long exposure). After 5 days in the absence of Dox, cells expressed higher levels of EGFP than those observed on the first day after Dox removal, but still lower levels than those cultured continuously without Dox. Scale bars = 200 µm (A, B) and 100 µm (C). Abbreviations: Dox, doxycycline; –Dox, without doxycycline in the medium; +Dox, with doxycycline in the medium; EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell.

View larger version (32K): [in this window] [in a new window]   Figure 3. Dox-responsive EGFP expression in EBs derived from the double-transfected ESC clone. (A): ESC-specific and differentiation marker genes examined in undifferentiated ESC clone (#J-d) and 12-day-old EBs by RT-PCR. The 12-day-old EBs were cultured in the presence or absence of Dox (1 ng/ml). Oct-3/4 and Nanog were used as ESC-specific marker genes. AFP and albumin (for endoderm), -MHC and brachyury (for mesoderm), ND-1 and NF68kD (for ectoderm), and Cdx2 and CG- (for trophectoderm) were used as differentiation marker genes. GAPDH was used as an internal control. The EBs showed downregulation of the ESC-specific marker genes and upregulation of the differentiation marker genes. EGFP mRNA expression was suppressed by treatment with Dox. (B): EGFP expression in the EBs was gradually downregulated after treatment with 1 ng/ml Dox for 5 days, becoming almost undetectable by 12 days under a fluorescence microscope. (C): The slight EGFP expression was still observed inside of the EBs. (D): The 12-day-old EBs were plated and treated with or without Dox. Cells differentiated into a variety of cell types including derivatives of endoderm (AFP), mesoderm (Desmin), and ectoderm (NCAM) by immunostaining displayed Dox-responsive EGFP expression and almost homogeneous tTA expression. Scale bars = 100 µm (B–D). Abbreviations: AFP, -fetoprotein; -MHC, -myosin heavy chain; CG-, chorionic gonadotropin-; CMK6, monkey ESCs (wild-type); DAPI, 4',6-diamidino-2-phenylindole; Dox, doxycycline; –Dox, without doxycycline in the medium; +Dox, with doxycycline in the medium; EB, embryoid body; EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEF, mouse embryonic fibroblast; NCAM, neural cell adhesion molecule; ND-1, neurogenic differentiation-1; NF68kD, neurofilament 68 kDa; RT-PCR, reverse transcription-polymerase chain reaction; tTA, tetracycline-controlled transactivator; VP16, herpes simplex virus.

View larger version (58K): [in this window] [in a new window]   Figure 4. Dox-responsive EGFP expression in teratomas derived from the double-transfected ESC clone. (A): Histological analysis of teratomas formed in untreated SCID mice or SCID mice treated with Dox (500 µg/ml) in drinking water for 60–70 days. The paraffin-embedded teratoma sections revealed by HE staining that the clone differentiated into cells of all three germ layers, including endodermal epithelium, cartilage, muscle, and neuroepithelium. (B): Dox-responsive EGFP expression in the teratomas was observed by fluorescence stereomicroscopy. Such teratomas exhibited the downregulation of EGFP expression after the addition of Dox in drinking water for 10.5 days before harvesting. (C, D): Whereas EGFP expression in the teratomas appeared to be rather heterogeneous (C), Dox-responsive EGFP expression and tTA expression in various types of cells could be observed (D). Scale bars = 100 µm (A, C, D). Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; Dox, doxycycline; –Dox, without doxycycline in the medium; +Dox, with doxycycline in the medium; EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; HE, hematoxylin and eosin; SCID, severe combined immunodeficient; tTA, tetracycline-controlled transactivator; VP16, herpes simplex virus.

	CONCLUSION

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This study was supported in part by grants from The National Bio-Resource Project, The Ministry of Education, Culture, Sports, Sciences, and Technology (MEXT) of Japan, and the Japan Society for the Promotion of Science.

	REFERENCES

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion Disclosures Acknowledgments References

 

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