Faculty Profiles - SERA Takashi

写真a

SERA Takashi

Organization

Faculty of Interdisciplinary Science and Engineering in Health Systems Professor

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Degree

Ph.D. in Engineering ( 1993.3 Kyoto University )

Papers

Necessity of Flanking Repeats R1′ and R8′ of Human Pumilio1 Protein for RNA Binding Reviewed

Kento Nakamura, Taishu Nakao, Tomoaki Mori, Serika Ohno, Yusuke Fujita, Keisuke Masaoka, Kazuki Sakabayashi, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

Biochemistry 60 ( 40 ) 3007 - 3015 2021.9

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Authorship：Last author,　Corresponding author Publishing type：Research paper (scientific journal) Publisher：American Chemical Society (ACS)

DOI： 10.1021/acs.biochem.1c00445

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Genome sequence analysis of new plum pox virus isolates from Japan Reviewed International journal

Tomoaki Mori, Chiaki Warner, Serika Ohno, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

BMC Research Notes 14 ( 1 ) 266 - 266 2021.7

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

<title>Abstract</title><sec>
<title>Objective</title>
To find mutations that may have recently occurred in <italic>Plum pox virus</italic> (PPV), we collected six PPV-infected plum/peach trees from the western part of Japan and one from the eastern part. After sequencing the full-length PPV genomic RNAs, we compared the amino acid sequences with representative isolates of each PPV strain.

</sec><sec>
<title>Results</title>
All new isolates were found to belong to the PPV-D strain: the six isolates collected from western Japan were identified as the West-Japan strain while the one collected from eastern Japan as the East-Japan strain. Amino acid sequence analysis of these seven isolates suggested that the 1407th and 1529th amino acid residues are characteristic of the West-Japan and the East-Japan strains, respectively. Comparing them with the corresponding amino acid residues of the 47 non-Japanese PPV-D isolates revealed that these amino acid residues are undoubtedly unique. A further examination of the relevant amino acid residues of the other 210 PPV-D isolates collected in Japan generated a new hypothesis regarding the invasion route from overseas and the subsequent diffusion route within Japan: a PPV-D strain might have invaded the western part of Japan from overseas and spread throughout Japan.

</sec>

DOI： 10.1186/s13104-021-05683-9

PubMed

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Other Link： https://link.springer.com/article/10.1186/s13104-021-05683-9/fulltext.html
Site-specific integration by recruitment of a complex of ΦC31 integrase and donor DNA to a target site by using a tandem, artificial zinc-finger protein Reviewed International journal

Tatsuhiko Sumikawa, Serika Ohno, Takeharu Watanabe, Ryo Yamamoto, Miyu Yamano, Tomoaki Mori, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

Biochemistry 57 ( 50 ) 6868 - 6877 2018.11

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society (ACS)

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated “Tandem AZP”). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA. Thereafter, an exogenously added integrase was expected to integrate the donor DNA into a specific site in the acceptor DNA (as it might be in the human genome). We demonstrated in vitro that in the presence of Tandem AZP, ΦC31 integrase selectively integrated a donor plasmid into a target acceptor plasmid not only at 30 °C (the optimum temperature of the integrase) but also at 37 °C (for future application in humans). We expect that with further improvement of our current system, a combination of Tandem AZP with integrase/recombinase will enable site-specific integration in mammalian cells and provide safer gene therapy technology.

DOI： 10.1021/acs.biochem.8b00979

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Targeted silencing of SOX2 by an artificial transcription factor showed antitumor effect in lung and esophageal squamous cell carcinoma. Reviewed International journal

Etsuko Yokota, Tomoki Yamatsuji, Munenori Takaoka, Minoru Haisa, Nagio Takigawa, Noriko Miyake, Tomoko Ikeda, Tomoaki Mori, Serika Ohno, Takashi Sera, Takuya Fukazawa, Yoshio Naomoto

Oncotarget 8 ( 61 ) 103063 - 103076 2017.1

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Language：English Publishing type：Research paper (scientific journal)

SOX2 is a transcription factor essential for early mammalian development and for the maintenance of stem cells. Recently, SOX2 was identified as a lineage specific oncogene, recurrently amplified and activated in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger-based artificial transcription factor (ATF) to selectively suppress SOX2 expression in cancer cells and termed the system ATF/SOX2. We engineered the ATF using six zinc finger arrays designed to target a 19 bp site in the SOX2 distal promoter and a KOX transcriptional repressor domain. A recombinant adenoviral vector Ad-ATF/SOX2 that expresses ATF/SOX2 suppressed SOX2 at the mRNA and protein levels in lung and esophageal SCC cells expressing SOX2. In these kinds of cells, Ad-ATF/SOX2 decreased cell proliferation and colony formation more effectively than the recombinant adenoviral vector Ad-shSOX2, which expresses SOX2 short hairpin RNA (shSOX2). Ad-ATF/SOX2 induced the cell cycle inhibitor CDKN1A more strongly than Ad-shSOX2. Importantly, the ATF did not suppress the cell viability of normal human cells. Moreover, Ad-ATF/SOX2 effectively inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that ATF/SOX2 would lead to the development of an effective molecular-targeted therapy for lung and esophageal SCC.

DOI： 10.18632/oncotarget.21523

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Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein-staphylococcal nuclease hybrid Reviewed International journal

Tomoaki Mori, Kento Nakamura, Keisuke Masaoka, Yusuke Fujita, Ryosuke Morisada, Koichi Mori, Takamasa Tobimatsu, Takashi Sera

Biochemical and Biophysical Research Communications 479 ( 4 ) 736 - 740 2016.9

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier BV

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.

DOI： 10.1016/j.bbrc.2016.09.142

Web of Science

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Gene- and Protein-Delivered Zinc Finger–Staphylococcal Nuclease Hybrid for Inhibition of DNA Replication of Human Papillomavirus Reviewed

Takashi Mino, Tomoaki Mori, Yasuhiro Aoyama, Takashi Sera

PLoS ONE 8 ( 2 ) e56633 - e56633 2013.2

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Public Library of Science (PLoS)

DOI： 10.1371/journal.pone.0056633

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Enhanced Cleavage of Double-Stranded DNA by Artificial Zinc-Finger Nuclease Sandwiched between Two Zinc-Finger Proteins Reviewed

Yusuke Mineta, Tomoyuki Okamoto, Kosuke Takenaka, Norio Doi, Yasuhiro Aoyama, Takashi Sera

Biochemistry 47 12257 - 12259 2008.11

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Chemical Society (ACS)

DOI： 10.1021/bi801800k

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Modulation of endogenous VEGF-A expression by artificial transcription factors.

Tomoaki Mori, Jun Sasaki, Yasuhiro Aoyama, Takashi Sera

Nucleic acids symposium series (2004) ( 51 ) 345 - 346 2007

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The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To modulate the endogenous gene expression artificially, we previously designed a six-finger AZP to recognize a 19-bp DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a repressor domain to generate an artificial transcription factor (ATF). Using the ATF, we demonstrated efficient modulation of the VEGF-A expression. In the present study, we evaluate the ability of the ATF to modulate the gene expression in more detail. First we examined the ability of the ATF under hypoxia condition. ELISA of the VEGF-A protein in the culture medium revealed that the gene-delivered ATF repressed the expression rate of the VEGF-A gene under hypoxia condition.

DOI： 10.1093/nass/nrm173

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Development of protein-based antiviral drugs for human papillomaviruses.

Takashi Mino, Tomoaki Mori, Yasuhiro Aoyama, Takashi Sera

Nucleic acids symposium series (2004) ( 51 ) 427 - 428 2007

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Recently, we designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined another approach to delivery of AZPs. We constructed cell-permeable AZPs by fusing an AZP previously generated for inhibition of HPV-18 DNA replication with a cell-penetrating peptide (CPP), and confirmed that these CPP-AZP fusions reduced the replication rate in transient replication assays when added to the culture medium. In particular, 250 nM CPP-AZP reduced HPV-18 DNA replication to 3% of that of a control experiment. Western blot analysis detected 7% of the CPP-AZP added to the culture medium in the cell lysates, and demonstrated that greater internalization of CPP-AZP into mammalian cells causes greater inhibition of viral DNA replication. Furthermore, CPP-AZP did not show any significant cytotoxixity in MTT assays. Thus, our results demonstrate that cell-permeable AZPs could serve as potent protein-based antiviral drugs.

DOI： 10.1093/nass/nrm214

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Inhibition of DNA replication of human papillomavirus by artificial zinc finger proteins. Reviewed International journal

Takashi Mino, Takeaki Hatono, Naoki Matsumoto, Tomoaki Mori, Yusuke Mineta, Yasuhiro Aoyama, Takashi Sera

Journal of virology 80 ( 11 ) 5405 - 5412 2006.6

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to the inhibition of replication of a mammalian DNA virus, human papillomavirus type 18 (HPV-18). Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed by using our nondegenerate recognition code table and were constructed to block binding of the HPV-18 E2 replication protein to the replication origin. Both of the newly designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and they efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited viral DNA replication, especially AZP(HPV)-2, which reduced the replication level to approximately 10%. We also demonstrated in transient replication assays, using plasmids with mutant replication origins, that AZP(HPV)-2 could precisely recognize the replication origin in mammalian cells. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses but also to mammalian DNA viruses.

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Inhibition of Virus DNA Replication by Artificial Zinc Finger Proteins Reviewed

Takashi Sera

Journal of Virology 79 ( 4 ) 2614 - 2619 2005.2

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Authorship：Lead author,　Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

ABSTRACT

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of theBeet severe curly top virus(BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenicArabidopsisplants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.

DOI： 10.1128/jvi.79.4.2614-2619.2005

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Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins Reviewed

Kiyoshi Tachikawa, Oliver Schröder, Gerhard Frey, Steven P. Briggs, Takashi Sera

Proceedings of the National Academy of Sciences 101 ( 42 ) 15225 - 15230 2004.10

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Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences

We describe a facile method to activate or repress transcription of endogenous genes in a quantitative and specific manner by treatment with designed regulatory proteins (DRPs), in which artificial transcription factors (ATFs) are fused to cell-penetrating peptides (CPPs). Penetration of DRPs into cells is mediated by an N-terminal CPP fused to a nuclear localization signal; a DNA-binding domain and a transactivation domain follow. The DNA-binding domain was targeted to the vascular endothelial growth factor (VEGF)-A gene. An agonist DRP was rapidly taken up by cells and transported to the nucleus; soon after, the cells began transcribing the gene and secreting VEGF-A protein in a dose-dependent manner. Multiple copies of a short oligopeptide derived from a minimal transactivation domain of human β-catenin was stronger than VP-16. The SRDX domain from the plant transcription factor, SUPERMAN, changed the DRP to a hypoxia-induced antagonist of VEGF-A. DRPs combine many of the potential benefits of transgenes with those of recombinant proteins.

DOI： 10.1073/pnas.0406473101

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Research Projects

新規人工RNA結合タンパク質の革新的デザイン法の開発とその有効性の検証

Grant number：22H02200 2022.04 - 2025.03

日本学術振興会科学研究費助成事業基盤研究(B)

世良貴史

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Grant amount：\17420000 （ Direct expense: \13400000 、 Indirect expense：\4020000 ）

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Development of innovative design method for new artificial RNA-binding proteins

Grant number：19H02834 2019.04 - 2022.03

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

Sera Takashi

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Grant amount：\17290000 （ Direct expense: \13300000 、 Indirect expense：\3990000 ）

To develop artificial RNA-binding proteins, we focused on the R1'/ R8'domains at the N- and C-termini of the human Pumilio protein (hPUM), whose involvement in RNA recognition was unclear, and clarified their functions related to RNA binding. By preparing various variants of the domain and comparing their RNA binding characteristics with the wild type, we found that these domains, which do not directly interact with the base of the target RNA, makes an important contribution to RNA recognition of hPUM by stabilizing the entire protein structure.

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Development of a site-specific gene integration method to human genomes

Grant number：16K14633 2016.04 - 2019.03

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

Sera Takashi

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Grant amount：\3900000 （ Direct expense: \3000000 、 Indirect expense：\900000 ）

In order to overcome the problems of conventional gene therapy, we aimed to develop a new technology that enables site-specific gene insertion by making an integrase spatially close to the target DNA site. To this end, a new artificial DNA binding protein is created by connecting two kinds of artificial DNA binding proteins that specifically bind to their own target sites respectively with a flexible peptide linker, and using this novel protein, in the in vitro model system, we succeeded in specifically inserting another DNA into the target DNA.

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Development of artificial transcription factors for the treatment of lung and esophageal squamous cell carcinoma

Grant number：26462141 2014.04 - 2017.03

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

Naomoto Yoshio

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Grant amount：\4940000 （ Direct expense: \3800000 、 Indirect expense：\1140000 ）

Sox2 was identified as a lineage specific oncogene, recurrently amplified in lung and esophageal squamous cell carcinoma (SCC). In this study, we have developed a zinc finger based artificial transcription factor (ATF) to suppress Sox2 expression in cancer cells and termed the system ATF/Sox2. A transient transfection reporter assay demonstrated that ATF/Sox2 repressed Sox2 transcriptional activity in Sox2 expressing lung and esophageal SCC cells. A recombinant adenoviral vector: Ad-ATF/Sox2 that expresses ATF/Sox2 suppressed Sox2 at the mRNA and protein levels in lung and esophageal SCC cells. Importantly, in these cells, Ad-ATF/Sox2 decreased cell proliferation and colony formation. Moreover, Ad-ATF/SOX2 significantly inhibited tumor growth in a lung SCC xenograft mouse model. These results indicate that Sox2 silencing by ATF/ Sox2 could lead to the development of effective molecular-targeted therapies for lung and esophageal SCC.

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Generation of plants resistant to DNA viruses using artificial DNA-binding proteins

Grant number：25292036 2013.04 - 2016.03

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

SERA TAKASHI

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Grant amount：\18720000 （ Direct expense: \14400000 、 Indirect expense：\4320000 ）

The goal of this study is to develop plants resistant to DNA viruses by inactivating them with artificial DNA-binding proteins. For this purpose, we designed an artificial DNA-binding protein to inactivate DNA virus of our interest and introduced the gene encoding the protein into plants, resulting in generation of the transgenic plants. Furthermore, we constructed an infection system based on an agroinoculation method to evaluate their virus resistance.

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Development of a global gene identification method

Grant number：24655155 2012.04 - 2015.03

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

SERA TAKASHI

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Grant amount：\4030000 （ Direct expense: \3100000 、 Indirect expense：\930000 ）

The goal of this study is to develop a method for a global gene identification. For this purpose, we designed nine molecules that recognized one specific target site of the gene of our interest, respectively, and generated them. Among them, we identified the best sample to modulate the expression of the target gene most effectively. Furthermore, we examined how to generate the phenotype of our purpose, and raised the induction efficiency to 60-70%.

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Studies of action mechanisms of radical enzyme systems for providing new paradigms of enzyme researches

Grant number：22570143 2010 - 2012

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

TORAYA Tetsuo, TOBIMATSU Takamasa, MORI Koichi, SERA Takashi, YOSHIZAWA Kazunari, SHIBATA Naoki

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Grant amount：\3770000 （ Direct expense: \2900000 、 Indirect expense：\870000 ）

Where should enzyme sciences be directed? I believe that
(1) microbe-inspired system enzymology and
(2)理論化学との連携による酵素学の非経験的学問化、が今後の酵素研究に重要なパラダイムであると考えて、ラジカル酵素システムを研究対象として、これらの方向への展開を目指した。
In this research project, our investigations of radical enzymes and their (re)activating proteins using the methodologies of biochemistry, genetic engineering, biophysics, X-ray crystallography, and theoretical chemistry opened a new horizon on enzyme sciences.

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Improvement of production efficiency of iPS cells

Grant number：22655052 2010 - 2011

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

SERA Takashi

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Grant amount：\3460000 （ Direct expense: \3100000 、 Indirect expense：\360000 ）

The goal of this study is to develop a new method to improve the production efficiency of iPS cells. We designed four molecules for the purpose. We generated and purified them. We identified the best molecule based on molecular biological properties. We furthermore improved the function of the molecule. Using it, we modulated the target function in cells.

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人工転写因子ライブラリーを用いた網羅的遺伝子同定

Grant number：18018025 2006 - 2007

日本学術振興会科学研究費助成事業特定領域研究

世良貴史

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Grant amount：\7900000 （ Direct expense: \7900000 ）

これまでに狙い通りの人工転写因子ができることを確認したが、作製したDNA結合部位の、人工転写因子発現ベクター用の改良型プリカーサーへのクローニング効率が現在のところ芳しくない。ライゲーションサンプルの大腸菌への形質転換後得られる形質転換体数がそれほど高くなく、今のところ実験に必要な水準を満たしていない。各メーカーのエレクトロコンピテントセルを試してみたが、市販品のエレクトロコンピテントセルでは満足のいく結果が得られなかった。そこでエレクトロコンピテントセルを自作して、多量の形質転換体を得ることを試みた。また得られたベクターの動物細胞への遺伝子導入を行った。すでに報告されている方法に沿って、動物細胞への遺伝子導入を試みたが、その効率は報告されているものより予想以上に低く、様々な導入条件を検討したが、満足のいくものではなかった。そこで各種メーカーの遺伝子導入試薬を用いて、様々な条件の検討を行ったが、5%以上の遺伝子導入効率を得ることはできなかった。また、従来のエレクトロポーレーション法によっても導入効率の改善は見られなかった。しかし、最近報告された新しい方法に基づくエレクトロポーレーション法を用いると、遺伝子導入を大幅に改善することができた。遺伝子導入に用いるパルス電圧、パルスを掛ける時間および回数等の各種条件を検討し、最終的に再現性よく50%以上の効率で遺伝子導入できることを見出した。

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A novel approach to inhibition of human virus infection : inhibition of DNA virus replication by artificial DNA-binding proteins

Grant number：17550154 2005 - 2006

Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

TAKAHASHI Sera

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Grant amount：\3600000 （ Direct expense: \3600000 ）

Recently, we demonstrated that plant DNA virus replication was inhibited in planta by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic plants resistant to DNA virus infection. Here we apply the AZP technology to inhibition of replication of a mammalian DNA virus. Two AZPs, designated AZP-1 and AZP-2, were designed using our nondegenerated recognition code table and constructed to block binding of a viral replication protein to its replication origin. Both the newly designed AZPs had much higher affinities towards the replication origin than the viral replication protein, and efficiently blocked the binding in vitro. Next, we established a transient replication assay to evaluate the ability of AZPs to inhibit virus replication. In the transient replication assays, both AZPs inhibited the viral DNA replication : AZP-2, especially, reduced the replication level to approximately 10%. We also demonstrated it in transient replication assays with mutant replication origins that AZP_<HPV>-2 could precisely recognize the replication origin in mammalian cells, thereby inhibiting virus replication. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses, but also to mammalian DNA viruses.

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人工転写因子を用いたがん増殖シグナルの人為的操作

Grant number：17012013 2005

日本学術振興会科学研究費助成事業特定領域研究

世良貴史

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Grant amount：\8500000 （ Direct expense: \8500000 ）

ヒトVEGFプロモーターを認識する人工DNA結合タンパク質に、種々の転写抑制ドメインをつないだ人工転写因子遺伝子をCMVプロモーターの下流に導入したヒト細胞発現ベクターを作成した。さらに、VEGFプロモーターの下流にルシフェラーゼ遺伝子を連結させたルシフェラーゼ・リポーターを作成し、ヒト由来細胞HEK293を用いて、どの人工転写因子(あるいは転写抑制ドメイン)がルシフェラーゼ発現量を最も抑制するかを定量できる系を確立した。このアッセイ系を用いて、KRAB転写抑制ドメインと同等もしくはそれ以上の活性を有するドメインを複数得ることに成功した。現在、その再現性を慎重に調べているところである。また、内在性のVEGF遺伝子の発現量をそのタンパク質生成量に基づいて定量できる系も確立した。
また、同時に人工転写因子タンパク質そのものの細胞導入によるVEGF発現抑制も試みた。現在、上述したように最も効果的な抑制ドメインの探索を行なっているので、まずKRAB転写抑制ドメインを有する人工転写因子に種々の細胞膜透過ペプチドを繋げたタンパク質の大腸菌発現ベクターを作成した。このうち、細胞膜透過ペプチドPTD-4を連結した人工転写因子タンパク質を大腸菌で過剰発現させ、その精製を完了した。上述したルシフェラーゼ・リポーター・アッセイ系において、このタンパク質導入により、同一タンパク質非存在下のルシフェラーゼ遺伝子発現量の10%にまで抑制できることを確認した。

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人工転写因子ライブラリーを用いた網羅的遺伝子同定

Grant number：17019036 2005

日本学術振興会科学研究費助成事業特定領域研究

世良貴史

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Grant amount：\4700000 （ Direct expense: \4700000 ）

まず、人工転写因子ライブラリー発現ベクターのプリカーサーを作成した。このベクターに人工転写因子遺伝子を導入した発現ベクターを作成し、このベクターでヒト由来細胞HEK293を形質転換し、この遺伝子が効率よく発現されることをウェスタンブロットで確認した。また、人工転写因子ライブラリー作成に必要なすべてのDNAフラグメントのデザインを完了した。まずグアニン(G)が豊富な塩基配列を認識できるように(ヒトのプロモーター配列はGが豊富な場合が多い)、ライブラリー全体の4分の1に該当するDNAフラグメント作成用のDNAオリゴマーを購入し、(部分的な)ライブラリーを作成した。このライブラリーを発現ベクターのプリカーサーに導入したもので大腸菌を形質転換し、生成してきた50個以上のコロニーからDNAベクターを単離し、各々の塩基配列を決定したところ、各DNAフラグメントがランダムにアッセンブルされて、狙い通りの(部分的だが)人工転写因子ライブラリーができることを確認した。こうして得られた人工転写因子ライブラリーをヒト由来細胞に遺伝子導入したが、1回目のセレクション操作における標的の性質を示した細胞の割合が極端に低かったので、現在、遺伝子導入時の実験条件等の最適化を行なっているところである。また、残り4分の3のDNAフラグメント作成用のDNAオリゴマーを購入し、完全なライブラリーの作成も現在行なっているところである。

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人工転写因子ライブラリーを用いた網羅的遺伝子同定

Grant number：16012230 2004

日本学術振興会科学研究費助成事業特定領域研究

世良貴史

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Grant amount：\5400000 （ Direct expense: \5400000 ）

まず、人工転写因子ライブラリー発現ベクターのプリカーサーを作成した。このベクターに人工転写因子遺伝子を導入した発現ベクターを作成し、このベクターでヒト由来細胞HEK293を形質転換し、この遺伝子が効率よく発現されることをウェスタンブロットで確認した。また、人工転写因子ライブラリー作成に必要な768本のDNAフラグメントのデザインを完了した。しかし、実際に支給された研究費ではライブラリー作成に必要なすべてのDNAオリゴマーを購入できないので、まずグアニン(G)が豊富な塩基配列を認識できるように(ヒトのプロモーター配列はGが豊富な場合が多い)、全体の4分の1の192本分のDNAフラグメント作成用のDNAオリゴマーを購入し、(部分的な)ジンク・フィンガー・ドメイン・ライブラリーを作成した。このライブラリーを発現ベクターのプリカーサーに導入したもので大腸菌を形質転換し、生成してきた50個以上のコロニーからDNAベクターを単離し、各々の塩基配列を決定したところ、フィンガー・ドメインがランダムにアッセンブルされて、狙い通りの(部分的だが)人工転写因子ライブラリーができることを確認した。こうして得られた人工転写因子ライブラリーをヒト由来細胞に遺伝子導入したが、1回目のセレクション操作における標的の形質を示した細胞の割合が極端に低かったので、現在、遺伝子導入時の実験条件の最適化を行なっているところである。

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