JP5110580B2 - Lymphatic vessel visualization transgenic medaka and screening method for lymphangiogenesis inhibitor using the medaka - Google Patents
Lymphatic vessel visualization transgenic medaka and screening method for lymphangiogenesis inhibitor using the medaka Download PDFInfo
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Description
本発明は、リンパ管可視化トランスジェニックメダカ、該メダカを作製するためのプロモーター、ベクター、形質転換細胞及び該トランスジェニックメダカを用いたリンパ管新生阻害薬のスクリーニング法に関する。 The present invention relates to a lymph vessel visualization transgenic medaka, a promoter, a vector, a transformed cell for producing the medaka, and a method for screening a lymphangiogenesis inhibitor using the transgenic medaka.
癌の治療を困難にしている原因の一つにリンパ節転移がある。癌細胞は腫瘍内あるいは腫瘍周囲に新生リンパ管を誘導し、結果として形成された豊富なリンパ管は、癌細胞が侵入する機会を増やし、癌のリンパ節転移を促進することが分かっている。そこで、抗がん剤の新たな標的としてリンパ管新生の阻害が着目され開発が進められている。しかし、リンパ管新生の機構については、「VEGF-Cが主に静脈の内皮細胞に由来するリンパ管前駆細胞に発現しているVEGFR-3に受容され、VEGFR-3がリン酸化シグナルを発信し何らかの経路で転写因子Prox-1を発現させリンパ管を新生させる」ということ以外はっきりしていない。そのため、リンパ管新生阻害剤の探索方法としては専ら培養細胞系を用いVEGFR-3のリ
ン酸化を阻害する効果を調べるという手法以外確立されていない。
本発明は、リンパ管新生に影響する物質のスクリーニング方法、該スクリーニング方法に適用するのに有用なトランスジェニックメダカを提供することを目的とする。 An object of the present invention is to provide a method for screening a substance that affects lymphangiogenesis, and a transgenic medaka useful for application to the screening method.
本発明者は、上記課題に鑑み検討を重ねた結果、メダカリンパ系に発現するVascular endothelial growth factor receptor 3(VEGFR-3)遺伝子のプロモーター領域を含むDNA
断片を同定した。そして、実際にこの断片がメダカ個体内でリンパ管細胞に選択的にレポーター遺伝子を発現させ得ることを実証した。
As a result of repeated examinations in view of the above problems, the present inventor has obtained DNA containing the promoter region of Vascular endothelial growth factor receptor 3 (VEGFR-3) gene expressed in the medaka lymphatic system.
Fragments were identified. In fact, it was demonstrated that this fragment can selectively express a reporter gene in lymphatic cells in a medaka individual.
本発明は、リンパ管細胞において任意の遺伝子発現を有するプロモーター活性のあるDNA断片、それを含むプラスミドベクター、およびトランスジェニックメダカ、さらにリン
パ関係性を阻害する物質のスクリーニング法に関する。
項1. メダカ由来のVascular endothelial growth factor receptor 3(VEGFR-3)遺伝子のプロモーターの制御下にレポーター遺伝子を連結した遺伝子構築物ないし発現ベクターで形質転換されたトランスジェニックメダカ。
項2. 項1に記載のメダカに被験物質を作用させ、該被験物質のリンパ管の成長に対する影響を評価することを特徴とする、リンパ管新生阻害薬のスクリーニング法。
The present invention relates to a DNA fragment having promoter activity having an arbitrary gene expression in lymphatic cells, a plasmid vector containing the DNA fragment, a transgenic medaka, and a screening method for a substance that inhibits lymphatic relationship.
Item 1. A transgenic medaka transformed with a gene construct or expression vector in which a reporter gene is linked under the control of the promoter of medaka-derived Vascular endothelial growth factor receptor 3 (VEGFR-3) gene.
Item 2. A screening method for a lymphangiogenesis inhibitor, which comprises causing a test substance to act on the medaka according to Item 1, and evaluating the influence of the test substance on the growth of lymphatic vessels.
本発明により、メダカのリンパ管細胞で発現可能なメダカVEGFR-3プロモーターを初め
て取得できた。
According to the present invention, the medaka VEGFR-3 promoter that can be expressed in medaka lymphatic cells can be obtained for the first time.
本発明のプロモーターを用いることで、任意の異種遺伝子をメダカのリンパ管細胞で高発現することができ、トランスジェニックメダカを容易に得ることができるようになった。 By using the promoter of the present invention, any heterologous gene can be highly expressed in medaka lymphatic cells, and transgenic medaka can be easily obtained.
本発明のトランスジェニックメダカは稚魚は、リンパ管全体と血管(静脈)の一部におい
てレポーター遺伝子を発現するので、この稚魚を用いてリンパ管新生阻害薬をスクリーニングすることができる。例えばメダカとして透明なメダカを使用し、プロモーターにGFP
、YFPなどの蛍光蛋白質、ルシフェラーゼを含むレポーター遺伝子を連結することで、生
きた個体内の細胞を視覚的に観察することができ、薬物候補化合物を含む生理活性物質のリンパ系形成阻害作用をリアルタイムで観察することができる。このようにして得られたリンパ管新生阻害薬は抗がん剤としてだけでなく、リンパ管腫のようなリンパ管形成異常による疾患の治療薬としても使用できる可能性がある。
In the transgenic medaka of the present invention, juveniles express a reporter gene in the whole lymphatic vessels and in a part of blood vessels (veins), and therefore, lymphangiogenesis inhibitors can be screened using these juveniles. For example, a transparent medaka is used as a medaka and GFP is used as a promoter.
By linking a reporter gene containing fluorescent protein such as YFP and luciferase, cells in a living individual can be visually observed, and the lymphatic system formation inhibitory action of physiologically active substances including drug candidate compounds is real-time. Can be observed. The thus obtained lymphangiogenesis inhibitor may be used not only as an anticancer agent but also as a therapeutic agent for diseases caused by abnormal lymphangiogenesis such as lymphangioma.
レポーター遺伝子として蛍光タンパク質を組み込んだ本発明のトランスジェニックメダカは、蛍光顕微鏡で観察するだけで生きたままリンパ管を可視化することができる。このようなリンパ管可視化トランスジェニックメダカにリンパ管新生阻害薬の候補物質を投与し、そのトランスジェニックメダカのリンパ管新生を蛍光顕微鏡下で観察することで計測し、スクリーニングを行うことができる。 The transgenic medaka of the present invention in which a fluorescent protein is incorporated as a reporter gene can visualize lymphatic vessels while alive only by observation with a fluorescence microscope. A lymphatic angiogenesis inhibitor candidate substance is administered to such a lymphatic vessel visualization transgenic medaka, and lymphangiogenesis of the transgenic medaka can be measured and screened under a fluorescence microscope.
本発明者は、VEGFR-3がメダカのリンパ管前駆細胞に選択的に発現していると予測し、
そのプロモーターを取得した。VEGFR-3プロモーターにレポーター遺伝子を連結して導入
したトランスジェニックメダカにおいて、VEGFR-3は成熟メダカではリンパ管のみに発現
し、メダカの稚魚においては、静脈とリンパ管の両方に発現していた。この稚魚にVEGFR-3のリン酸化を阻害することが公知の3-(4-Dimethylamino-naphthalen-1-ylmethylene)-1,3-dihydro-indol-2-one(MAZ51;CALBIOCHEM社)を作用させると、リンパ管新生が選択的に阻害されることが確認された。
The present inventors predicted that VEGFR-3 is selectively expressed in medaka lymphatic progenitor cells,
The promoter was obtained. In transgenic medakas introduced with a reporter gene linked to the VEGFR-3 promoter, VEGFR-3 was expressed only in lymphatic vessels in mature medaka, and in both venous and lymphatic vessels in medaka fry. 3- (4-Dimethylamino-naphthalen-1-ylmethylene) -1,3-dihydro-indol-2-one (MAZ51; CALBIOCHEM), which is known to inhibit phosphorylation of VEGFR-3, acts on this fry It was confirmed that lymphangiogenesis was selectively inhibited.
このように、本発明で得たメダカVEGFR-3プロモーターとレポーター遺伝子を連結して
トランスジェニックメダカを作製することで、リンパ管新生を阻害する抗ガン剤をスクリーニングできることが、本発明の特徴である。
Thus, it is a feature of the present invention that an anti-cancer agent that inhibits lymphangiogenesis can be screened by linking the medaka VEGFR-3 promoter obtained in the present invention and a reporter gene to produce a transgenic medaka. .
メダカ由来のVEGFR-3プロモーターの配列を配列番号1に示す。 The sequence of the medaka-derived VEGFR-3 promoter is shown in SEQ ID NO: 1.
本明細書において、「メダカプロモーター」とは、メダカにおいてプロモーターとして作用する能力(プロモーター機能)を有するDNA断片のことである。「プロモーター」は
、魚類、特にメダカなどの小型硬骨魚類において遺伝子発現を促進する活性を有するDNA
断片を意味する。プロモーターは、DNAを鋳型としてmRNA合成酵素(転写)を開始するDNA上の特定塩基配列を意味する。具体的には、メダカ由来のVEGFR-3遺伝子のプロモーター
およびその改変体が挙げられる。該プロモーター改変体は、メダカなどの小型硬骨魚類(
特にメダカ)においてプロモーター活性を有するプロモーター改変体を意味し、具体的に
は、以下のものが挙げられる:
(a)配列番号1の塩基配列からなるDNA又はその相補鎖
(b)(a)の塩基配列において1もしくは複数の塩基配列が欠失、置換、挿入もしくは付加された塩基配列からなり、かつメダカプロモーターとして作用する能力を有するDNA
(c)(a)又は(b)の塩基配列からなるDNAとストリンジェントな条件下でハイブリダ
イズし、かつメダカプロモーターとして作用する能力を有するDNA。
In the present specification, the “medaka promoter” refers to a DNA fragment having the ability to act as a promoter (promoter function) in medaka. "Promoter" is a DNA that has the activity to promote gene expression in fish, especially small teleosts such as medaka
Means a fragment. A promoter means a specific base sequence on DNA that initiates mRNA synthase (transcription) using DNA as a template. Specific examples include a promoter of a medaka-derived VEGFR-3 gene and a modified form thereof. The modified promoter is a small teleost such as medaka (
In particular, it means a modified promoter having promoter activity in medaka), and specifically includes the following:
(A) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary strand (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted, inserted or added in the nucleotide sequence of (a), and medaka DNA with the ability to act as a promoter
(C) DNA having the ability to hybridize with a DNA comprising the nucleotide sequence of (a) or (b) under stringent conditions and to act as a medaka promoter.
ストリンジェントな条件としては、具体的には、6×SSC(0.9M NaCl,0.09M クエン酸三ナトリウム)または6×SSPE(3M NaCl,0,2M NaH2PO4,20mM EDTA・2Na,pH7.4)中42℃でハイブリダイズさせ、さらに42℃で0.5×SSCにより洗浄する条件が、本発明のストリンジェントな条件の1例として挙げられるが、これに限定されるものではない。 As stringent conditions, specifically, 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, 2 M NaH 2 PO 4 , 20 mM EDTA · 2Na, The conditions of hybridization at 42 ° C. in pH 7.4) and further washing with 0.5 × SSC at 42 ° C. are mentioned as an example of the stringent conditions of the present invention, but are not limited thereto. .
本発明の1つの実施形態におけるプロモーターは、配列番号1記載の塩基配列からなるD
NAにおいて、1もしくは複数のDNAが欠失、置換、挿入もしくは付加された塩基配列から
なり、かつ、メダカプロモーターとして作用する能力を有するDNAである。本発明のメダ
カプロモーターには、配列番号1記載の塩基配列の3’または5’末端あるいは内部に、翻訳効率を上げる塩基配列などを付加したものや、3’または5’末端をプロモーター活性が有る限り、欠失したものが含まれる。
In one embodiment of the present invention, the promoter is a D consisting of the base sequence set forth in SEQ ID NO: 1.
In NA, it is a DNA having a base sequence in which one or more DNAs are deleted, substituted, inserted or added, and having the ability to act as a medaka promoter. The medaka promoter of the present invention has a promoter sequence added at the 3 ′ or 5 ′ end or inside of the base sequence shown in SEQ ID NO: 1 or a base sequence that increases translation efficiency, or has a promoter activity at the 3 ′ or 5 ′ end. As long as it is deleted.
本発明の「ベクター」は、上記プロモーターをクローニングベクターあるいはプロモーターを欠損したレポーターベクターに導入したものであり、例えば大腸菌由来の一般的なクローニングベクターとしてpBluescriptII, レポーターとしてルシフェラーゼを持つpGL3 Basic (Promega社)、レポーターとして蛍光蛋白質を持つpEGFPベクター(Clontech社)などが挙げられる。 The “vector” of the present invention is obtained by introducing the above promoter into a cloning vector or a reporter vector lacking the promoter. For example, pGL3 Basic (Promega) having pBluescriptII as a general cloning vector derived from E. coli and luciferase as a reporter. And pEGFP vector (Clontech) having a fluorescent protein as a reporter.
ベクターはそれぞれの魚類の宿主細胞に応じて選択する。本発明のプロモーターをベクターに導入する方法は、通常の遺伝子をベクターに導入する方法に従う。 The vector is selected according to the host cell of each fish. The method for introducing the promoter of the present invention into a vector follows the method for introducing a normal gene into a vector.
本発明の「トランスジェニックメダカ」とは、上記ベクターにレポーター遺伝子を組み込み、メダカの受精卵に導入することで得ることができる。ベクターに代えて、本発明のプロモーターに異種遺伝子を発現可能に連結して遺伝子構築物を作製し、該遺伝子構築物を同様にメダカの受精卵に導入することでトランスジェニックメダカを得ることもできる。 The “transgenic medaka” of the present invention can be obtained by incorporating a reporter gene into the vector and introducing it into a fertilized egg of medaka. Instead of a vector, a transgenic medaka can be obtained by linking a heterologous gene to a promoter of the present invention so that the gene can be expressed, and introducing the gene construct into a fertilized egg of medaka in the same manner.
本発明で得られたトランスジェニックメダカは、寄託機関の寄託対象生物に含まれていないので、本出願人により分譲可能な状態に維持されている。 Since the transgenic medaka obtained in the present invention is not included in the deposit target organism of the depository institution, it is maintained in a state that can be distributed by the present applicant.
メダカとしては、特に限定されないが、メダカSTII系統が、本来メダカの持つ色素(黒色、白色、虹色)を欠損する透明メダカの系統で、個体内の細胞で発現するレポータータンパク質(特に蛍光蛋白質)のシグナルを容易に観察することができるために好ましい。 The medaka is not particularly limited, but the medaka STII strain is a transparent medaka strain that lacks the original medaka pigment (black, white, iridescent), and is a reporter protein (especially a fluorescent protein) expressed in cells within the individual. This is preferable because the signal can be easily observed.
本発明のベクターを宿主細胞に導入する形質転換法としては、エレクトロポレーション法、マイクロインジェクション法、リン酸カルシウム法、リポソームを用いる方法あるいはパーティクルガン法などを挙げることができる。 Examples of transformation methods for introducing the vector of the present invention into host cells include electroporation method, microinjection method, calcium phosphate method, method using liposome, particle gun method and the like.
本発明のプロモーターに必要なエレメントを決定し、必須のエレメント以外の部分を改変したプロモーターを作製することができる。 Elements necessary for the promoter of the present invention can be determined, and a promoter in which parts other than essential elements are modified can be prepared.
本発明のプロモーター上にあるエレメント部位を決定する方法としては、例えばJoseph
Sambrook,David W. Russell Molecular Cloning 第3版のChapter13に書かれているデリーション法やPCRを用いて長さを調節したプロモーターを作製し、メダカ細胞等に形質転
換後、目的の条件でのプロモーター活性をレポーター遺伝子を用いて調査する方法がある。さらに詳細にエレメント配列を決定する場合は、Joseph Sambrook,David W. Russell Molecular Cloning 第3版のChapter13に書かれているようにプロモーター上にDNA変異を
導入して、またはPCR法を利用した変異の導入により、上記と同様に、メダカ細胞等に形
質転換後、プロモーター活性を調査し、もっとも重要なエレメント配列を決定する方法が挙げられる。
As a method for determining the element site on the promoter of the present invention, for example, Joseph
Sambrook, David W. Russell Molecular Cloning Produced a length-regulated promoter using the deletion method or PCR described in Chapter 13 of the third edition, transformed into medaka cells, etc., and then under the desired conditions There is a method for investigating the activity using a reporter gene. When determining the element sequence in more detail, DNA mutation is introduced into the promoter as described in Chapter 13 of Joseph Sambrook, David W. Russell Molecular Cloning 3rd edition, or the mutation using PCR method is used. As described above, after introduction into medaka cells and the like by introduction, a method of investigating promoter activity and determining the most important element sequence can be mentioned.
本発明のプロモーターの改変は、プロモーターのエレメントをタンデムに接続するなどの種々の方法により行うことができ、活性が変化したプロモーターを得ることができる。 Modification of the promoter of the present invention can be performed by various methods such as connecting the elements of the promoter in tandem, and a promoter with altered activity can be obtained.
本発明のプロモ−ターに接続することが好ましいレポーター遺伝子としては、蛍光タンパク質(緑色蛍光蛋白質(GFP)、黄色蛍光蛋白質(YFP)、青色蛍光蛋白質(BFP)、赤
色蛍光蛋白質(RFP)など)、エクオリン、アルカリホスファターゼ、ルシフェラーゼ 、β−グルクロニダーゼ、β−ラクタマーゼ、β−ガラクトシダーゼ、アセトヒドロキシ酸シンターゼ、クロラムフェニコールアセチルトランスフェラーゼ、西洋ワサビペルオキシダーゼ、ノパリンシンターゼ、オクタピンシンターゼなどが挙げられるが、定性的測定を行なう場合には肉眼的な観察が容易である点で蛍光蛋白質、定量的測定を行う場合にはルシフェラーゼが好ましい。
Reporter genes that are preferably connected to the promoter of the present invention include fluorescent proteins (green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), etc.), Examples include aequorin, alkaline phosphatase, luciferase, β-glucuronidase, β-lactamase, β-galactosidase, acetohydroxyacid synthase, chloramphenicol acetyltransferase, horseradish peroxidase, nopaline synthase, octapin synthase, etc. Fluorescent proteins are preferred in that they are easily observed with the naked eye when measurement is performed, and luciferases are preferable when quantitative measurement is performed.
以下、本発明を実施例に従いより詳細に説明するが、本発明はこれら実施例に限定されない。
実施例1
(1) Ol VEGFR-3上流領域の取得
Vascular endothelial growth factor receptor 3(VEGFR-3、別名FMS-like tyrosine kinase 4, FLT-4)遺伝子のヒト、マウス及びゼブラフィッシュのアミノ酸配列データをNCBI遺伝子データベース(http://www.ncbi.nlm.nih.gov)から取得した。このうちゼブラフィッシュの配列データ(NM_130945)をクエリとし、メダカのホモログ(Ol VEGFR-3)
をメダカゲノムBLAST検索サービス(http://dolphin.lab.nig.ac.jp/medaka/srch_db/search_medaka_blastdb2.php?lang=eg&lmode=all, golw_scaffold Hd-rR (200506))を使用
し予測した。Ol VEGFR-3の翻訳開始点から上流3787 bpまでの範囲をプロモーター領域と
予測しPCRにより増幅した。増幅に使用したプライマーはPrimer3(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)を用いて設計した上流gcagtccgtcaatactgagg(配列番号2)と下流ttctggactggagatctggag(配列番号3)にそれぞれKpn IとNco Iの制限酵素サイトをつなげた上流ggtaccgcagtccgtcaatactgagg(配列番号4)と下流ccatggctccagatctccagtccagaa(配列番号5)からなる配列をoperon社に合成を依頼して作製した。メダカHd-rR系統(♂)よりゲノムDNAを調製し(Dneasy tissue kit, QIAGEN社)これを鋳型として、上記プライマーを用いてGenomicDNA-PCRを行い、ゲノム断片をクローニングした。PCR
はポリメラーゼとしてEx Taq(Takara社)を使用し反応溶液組成は標準のプロトコールで、サーマルサイクラーとして GeneAmp PCR System 9700(Applied Biosystems社) を使
用し94℃ 4分、(94℃ 30秒、65℃(1回ごとに1℃下げる) 30秒、72℃ 4分)を7回、(94℃ 30秒、58℃ 30秒、72℃ 4分)を28回、72℃ 5分の温度変化によって増幅した。PCR
産物をアガロースゲル電気泳動により分離したところ、目的のサイズのバンドを確認した。ゲルより反応産物を抽出・精製した(Mini-elute kit, QIAGEN社)。精製したPCR産物
をTA-cloning法によってpGEM-Teasyベクター(Promega社)にクローニングした。こうし
て作成されたプラスミドベクターをMini-prep kit (QIAGEN社)を用いて調製した。取得した遺伝子配列が予測したOl VEGFR-3上流領域であることを検証するために、精製したプラスミドベクターを鋳型とし、DyeTerminator法を用いてDNAシーケンシングを行なった(BigDyeTerminator, ABI3130, AppliedBiosystemns社)。以上により、3787 bpのOl VEGFR-3
プロモーターを取得した。以下、当該塩基配列のことをpFLT4-4.0kと呼ぶ。
EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, this invention is not limited to these Examples.
Example 1
(1) Acquisition of upstream region of Ol VEGFR-3
The amino acid sequence data of human, mouse and zebrafish of the Vascular endothelial growth factor receptor 3 (VEGFR-3, also known as FMS-like tyrosine kinase 4, FLT-4) gene is NCBI gene database (http: //www.ncbi.nlm. obtained from nih.gov). Among them, zebrafish sequence data (NM_130945) was used as a query, and medaka homolog (Ol VEGFR-3)
Was predicted using the medaka genome BLAST search service (http://dolphin.lab.nig.ac.jp/medaka/srch_db/search_medaka_blastdb2.php?lang=eg&lmode=all, golw_scaffold Hd-rR (200506)). The range from the translation start point of Ol VEGFR-3 to 3787 bp upstream was predicted as the promoter region and amplified by PCR. The primers used for amplification were upstream gcagtccgtcaatactgagg (SEQ ID NO: 2) and downstream ttctggactggagatctggag (SEQ ID NO: 3) designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) A sequence consisting of upstream ggtaccgcagtccgtcaatactgagg (SEQ ID NO: 4) and downstream ccatggctccagatctccagtccagaa (SEQ ID NO: 5), each of which was connected to the restriction enzyme sites of Kpn I and Nco I, was prepared by requesting the synthesis from Operan. Genomic DNA was prepared from the medaka Hd-rR strain (♂) (Dneasy tissue kit, QIAGEN). Using this as a template, genomic DNA-PCR was performed using the above primers, and the genomic fragment was cloned. PCR
Uses Ex Taq (Takara) as the polymerase and the reaction solution composition is a standard protocol. GeneAmp PCR System 9700 (Applied Biosystems) is used as the thermal cycler at 94 ° C for 4 minutes (94 ° C for 30 seconds, 65 ° C ( Amplified by a temperature change of 30 ° C, 72 ° C 4 minutes), 7 times (94 ° C 30 seconds, 58 ° C 30 seconds, 72 ° C 4 minutes) 28 times, 72 ° C 5 minutes . PCR
When the products were separated by agarose gel electrophoresis, a band of the desired size was confirmed. The reaction product was extracted and purified from the gel (Mini-elute kit, QIAGEN). The purified PCR product was cloned into pGEM-Teasy vector (Promega) by TA-cloning method. The plasmid vector thus prepared was prepared using a Mini-prep kit (QIAGEN). In order to verify that the obtained gene sequence is the predicted upstream region of Ol VEGFR-3, DNA sequencing was performed using the purified plasmid vector as a template and the DyeTerminator method (BigDyeTerminator, ABI3130, AppliedBiosystems). By the above, 3787 bp Ol VEGFR-3
A promoter was obtained. Hereinafter, this base sequence is referred to as pFLT4-4.0k.
(2) 導入遺伝子の作製
Ol VEGFR-3プロモーターの下流でレポーター遺伝子を発現するプラスミドベクターを以下のように作成した。緑色蛍光蛋白質EGFP(Clontech社)の蛋白質をコードする領域を含む配列をNcoI/XbaI部位で切断し、pGL3 Basicベクター(Promega社)のルシフェラーゼ
遺伝子と置換した。また、マルチクローニングサイト(MCS)の内部の制限酵素配列を欠
損させるために、最も5’側のKpnI部位、および3’側のNcoI部位に上記でクローニングしたpFLT4-4.0kをサブクローニングした。構築したOl VEGFR-3プロモーターの下流でEGFPレポーター遺伝子を発現するプラスミドベクターをpFLT4:EGFPと呼ぶ。この発現プラスミドベクターを大腸菌DH5αにトランスフォーメンションし、QIAprep Mini kit(QIAGEN社
)を用いて精製した。
(2) Production of transgene
A plasmid vector expressing a reporter gene downstream of the Ol VEGFR-3 promoter was prepared as follows. The sequence containing the region encoding the protein of green fluorescent protein EGFP (Clontech) was cleaved at the NcoI / XbaI site and replaced with the luciferase gene of pGL3 Basic vector (Promega). In addition, in order to delete the restriction enzyme sequence in the multicloning site (MCS), pFLT4-4.0k cloned above was subcloned into the 5 ′ KpnI site and the 3 ′ NcoI site. A plasmid vector expressing an EGFP reporter gene downstream of the constructed Ol VEGFR-3 promoter is called pFLT4: EGFP. This expression plasmid vector was transformed into E. coli DH5α and purified using a QIAprep Mini kit (QIAGEN).
(3) 遺伝子の導入
上記で作成した発現ベクターを用いて、トランスジェニックメダカ系統の作成と樹立を行なった。遺伝子を導入するメダカは透明メダカであるSTII系統を使用した。メダカSTII系統は本来メダカの持つ色素(黒色、白色、虹色)を欠損する透明メダカの系統で、個体内の細胞で発現する蛍光蛋白質のシグナルを容易に観察することができる実験用メダカである(名古屋大学・若松教授より分与)。親メダカは循環型水槽(IWAKI pomp社)において27℃、14時間の明期、10時間の暗期によるライトサイクル下で飼育した。性成熟したメダカSTIIをペアリングし、産卵したメダカ受精卵を採卵し、氷冷したハンクス液に入れ、発生の進行を一時的に止めた。卵塊を形成する付着糸を実体顕微鏡下でピンセットを用いて丁寧に取り除き、一個一個ばらばらにした。未受精および未成熟卵はこの時点で取り除いた。注入する発現ベクター溶液はVector DNA, 30ng/μl, K2PO4(pH8.0), 0.1M, フェノールレッド(Sigma社、cell culture grade), 0.02%)と調整した。ガラス電極(外径1.0 mmφ, 内径0.85 mmφ ハーバード社)に注入DNA溶液を充填し、気泡を取り除いた後、
マイクロインジェクター(フェムトジェット、エッペンドルフ社)に接続し、実体顕微鏡下(オリンパス社)で1−2細胞期のメダカ受精卵内の細胞に0.2 pl〜0.5 pl注入した。DNA注入の終わった受精卵はembryonic solution (0.03% Red Sea Salt溶液)に移し、28℃でインキュベーションした。発生の進んだ胚(G0)において注入した発現プラスミドベクターに由来する蛍光蛋白質のシグナルを実体顕微鏡下(MZ-FLIII,ライカ社)で観察し、
ディジタルCCDカメラ(DP70, オリンパス社)で記録した。一次スクリーニングとして、
個体群の中で蛍光蛋白質の発現が認められたものだけを抽出し、次世代のために飼育した。
(3) Introduction of gene A transgenic medaka line was created and established using the expression vector prepared above. The medaka into which the gene was introduced was an STII strain that was a transparent medaka. The Medaka STII line is a transparent medaka line that originally lacks the medaka pigment (black, white, rainbow), and is an experimental medaka that allows easy observation of fluorescent protein signals expressed in cells within an individual. (Shared by Professor Wakamatsu, Nagoya University). The parent medaka was reared in a circulating water tank (IWAKI pomp) under a light cycle at 27 ° C. with a light period of 14 hours and a dark period of 10 hours. Paired with sexually mature medaka STII, spawned medaka fertilized eggs were collected and placed in ice-cold Hanks solution to temporarily stop the development. The attached yarns forming the egg mass were carefully removed with tweezers under a stereomicroscope and separated one by one. Unfertilized and immature eggs were removed at this point. The expression vector solution to be injected was adjusted with Vector DNA, 30 ng / μl, K 2 PO 4 (pH 8.0), 0.1 M, phenol red (Sigma, cell culture grade), 0.02%). After filling the glass electrode (outer diameter 1.0 mmφ, inner diameter 0.85 mmφ Harvard) with the injected DNA solution and removing the bubbles,
It connected to the microinjector (Femtojet, Eppendorf), and 0.2 pl-0.5 pl was inject | poured into the cell in the 1-2 cell stage medaka fertilized egg under a stereomicroscope (Olympus). The fertilized egg after DNA injection was transferred to an embryonic solution (0.03% Red Sea Salt solution) and incubated at 28 ° C. The signal of the fluorescent protein derived from the expression plasmid vector injected in the advanced embryo (G0) was observed under a stereomicroscope (MZ-FLIII, Leica),
Recording was made with a digital CCD camera (DP70, Olympus). As primary screening,
Only those in which the expression of fluorescent protein was recognized in the population were extracted and reared for the next generation.
成長したG0個体を個別に野生型の相手と交配し、得られた受精卵(F1)を個別に採卵した。もし、交配したG0個体の生殖系列細胞のゲノムに注入した発現ベクターが挿入されていれば、二次スクリーニングとしてF1個体を蛍光顕微鏡下で観察することにより、ある割合で(1細胞期にゲノムに挿入された場合50%)蛍光蛋白質を発現する個体が得られるはずである。その結果、生殖系列を介したトランスジェニック個体を得ることができた。以下同様にF1トランスジェニック個体を飼育し、野生型と交配しF2系統を得た。得られたトランスジェニックメダカの稚魚を図1に示し、成魚を図2に示す。また、血球可視化トランスジェニックメダカ(独立行政法人 放射線医学総合研究所から分与)と
交配させたメダカの写真を図3に示す。図3に示されるように、血管とリンパ管は明確に相違しており、本発明のトランスジェニックメダカが、リンパ管を特異的に標識できることが明らかにされた。
The grown G0 individuals were individually crossed with wild-type partners, and the resulting fertilized eggs (F1) were individually collected. If the injected expression vector is inserted into the genome of the mating G0 individual germline cell, as a secondary screening, F1 individuals are observed under a fluorescence microscope at a certain rate (to the genome at one cell stage). Individuals that express the fluorescent protein (50% when inserted) should be obtained. As a result, transgenic individuals via the germ line could be obtained. Similarly, an F1 transgenic individual was bred and crossed with a wild type to obtain an F2 line. The resulting transgenic medaka larvae are shown in FIG. 1, and adult fish are shown in FIG. Fig. 3 shows a photograph of a medaka mated with a blood cell visualization transgenic medaka (distributed from the National Institute of Radiological Sciences). As shown in FIG. 3, the blood vessels and the lymphatic vessels are clearly different, and it was revealed that the transgenic medaka of the present invention can specifically label the lymphatic vessels.
(4) リンパ管新生阻害薬の候補物質の投与
VEGFR-3のリン酸化を阻害することが公知の3-(4-Dimethylamino-naphthalen-1-ylmethylene)
-1,3-dihydro-indol-2-one; MAZ51(CALBIOCHEM社)を使用した。これをメダカの飼育水
(0.03 % 海水塩水)に50 μMの終濃度になるように溶かす。MAZ51は疎水性の物質であるため、まずMAZ51をDMSOに10 mMの濃度になるように溶かした後、0.03 % 海水塩水で200倍希釈することで水溶液を作製した。この水溶液中で卵膜除去した受精後2〜3日目胚を3日間培養した。得られたトランスジェニックメダカは、MAZ51を作用させていないメダカ
と比較して、リンパ管の形成が阻害されていた(図4)。
(4) Administration of candidate substances for lymphangiogenesis inhibitors
3- (4-Dimethylamino-naphthalen-1-ylmethylene) known to inhibit phosphorylation of VEGFR-3
-1,3-dihydro-indol-2-one; MAZ51 (CALBIOCHEM) was used. Dissolve this in medaka breeding water (0.03% seawater brine) to a final concentration of 50 μM. Since MAZ51 is a hydrophobic substance, MAZ51 was first dissolved in DMSO to a concentration of 10 mM, and then diluted 200-fold with 0.03% seawater brine to prepare an aqueous solution. The embryos 2 to 3 days after fertilization after removal of the egg membrane in this aqueous solution were cultured for 3 days. In the obtained transgenic medaka, the formation of lymphatic vessels was inhibited as compared with the medaka not treated with MAZ51 (FIG. 4).
(5) リンパ管新生阻害薬の候補物質の評価
この胚のリンパ管の形成結果を蛍光実体顕微鏡、共焦点レーザ顕微鏡などを使用し蛍光観察し、その画像データを通常のものと比較することでリンパ管形成阻害効果を評価することができる。また、このトランスジェニックメダカは発生期においては血管にもGFPを
発現しているため血管新生を阻害することを目的とした抗がん剤の効果を評価することも可能である。
(5) Evaluation of candidate substances for lymphangiogenesis inhibitors The results of the formation of lymph vessels in this embryo are observed using a fluorescent stereomicroscope, confocal laser microscope, etc., and the image data is compared with the usual one. The effect of inhibiting lymphangiogenesis can be evaluated. In addition, since this transgenic medaka also expresses GFP in blood vessels in the developmental stage, it is also possible to evaluate the effects of anticancer agents aimed at inhibiting angiogenesis.
Claims (2)
(a)配列番号1の塩基配列からなるDNA又はその相補鎖
(b)(a)の塩基配列において1もしくは複数の塩基配列が欠失、置換、挿入もしくは付加された塩基配列からなり、かつメダカプロモーターとして作用する能力を有するDNA
のいずれかである、トランスジェニックメダカ。 It is transformed with a gene construct or expression vector in which a reporter gene is linked under the control of a medaka-derived Vascular endothelial growth factor receptor 3 (VEGFR-3) gene promoter. (b):
(A) DNA comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary strand (b) consisting of a nucleotide sequence in which one or more nucleotide sequences are deleted, substituted, inserted or added in the nucleotide sequence of (a), and medaka DNA with the ability to act as a promoter
A transgenic medaka that is either
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