JP2006101828A - Both genes encoding thymus organogenesis factor 1(tof1) and thymus organogenesis factor 2(tof2) and use of the genes - Google Patents

Both genes encoding thymus organogenesis factor 1(tof1) and thymus organogenesis factor 2(tof2) and use of the genes Download PDF

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JP2006101828A
JP2006101828A JP2004296317A JP2004296317A JP2006101828A JP 2006101828 A JP2006101828 A JP 2006101828A JP 2004296317 A JP2004296317 A JP 2004296317A JP 2004296317 A JP2004296317 A JP 2004296317A JP 2006101828 A JP2006101828 A JP 2006101828A
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tof1
thymus
genes
tof2
base sequence
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Yosuke Takahama
洋介 高濱
Shuhei Tomita
修平 冨田
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University of Tokushima NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that the elucidation of thymus organogenesis mechanism has very slowly progressed yet, despite the fact that the thymus is a central organ for a biophylaxis or immune system against infectious diseases, cancer, and the like, and such a TOF1 or a gene encoding the same has considerably contributed as a means for diagnosing, treating, preventing, or the like, of immunological diseases. <P>SOLUTION: The thymus organogenesis factors TOF1 and TOF2 and genes respectively encoding these factors are provided. A primer for PCR (polymerase chain reaction), a probe for hybridization, and the like, are also provided, respectively. These substances are useful as curatives, diagnoses, clinical examinations, prevention, and the like, for various and diversified immune diseases caused by thymus gland disorders. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

この発明は、新規な胸腺器官発生因子ＴＯＦ１及びＴＯＦ２両遺伝子とその用途に関するものである。   The present invention relates to a novel gene for both thymus organ development factors TOF1 and TOF2 and uses thereof.

胸腺は、細胞障害性Ｔリンパ球、ヘルパーＴ細胞、サプレッサーＴ細胞等を活発に産生することにより、感染症や癌等に対する生体防御あるいは免疫システムにおいて中枢的機能を演じる器官であることが知られており、骨髄と共に第一次リンパ性器官と呼ばれている。また、胸腺発生関連遺伝子として、ＦｏｘＮ１（非特許文献１）、Ｈｏｘａ３（非特許文献２）、Ｐａｘ１（非特許文献３）等が公知である。更に、胸腺器官発生の初期に発現される因子として、新規な免疫グロブリンスーパファミリーに属するＥＶＡとその遺伝子が報告されてはいる（非特許文献４）。しかし、胸腺器官発生因子とその利用に関する特許文献は見当たらず、胸腺上皮プロゼニター細胞とその用途に係る技術が知られているに過ぎない（特許文献１及び２）。
ＷＯ０２／０５１９８８． ＷＯ０３／１０５８７４． Ｎａｔｕｒｅ，３７２（６５０１），１０３−１０７，１９９４． Ｄｅｖｅｌｐｍｅｎｔａｌ Ｂｉｏｌｏｇｙ，１９５（１），１９５（１）， １−１５，１９９８． Ｊｏｕｒｎａｌ ｏｆ Ｉｍｍｕｎｏｌｏｇｙ，１６４（１１），５７５３−５７６０，２０００． Ｊｏｕｒｎａｌ ｏｆ Ｃｅｌｌ Ｂｉｏｌｏｇｙ，１４１（４），１０６１−１０７１，１９９８． The thymus is known to be an organ that plays a central role in the defense of the body or immune system against infectious diseases and cancer by actively producing cytotoxic T lymphocytes, helper T cells, suppressor T cells, etc. It is called the primary lymphoid organ along with the bone marrow. Further, FoxN1 (Non-patent document 1), Hoxa3 (Non-patent document 2), Pax1 (Non-patent document 3) and the like are known as thymic development-related genes. Furthermore, as a factor expressed in the early stage of thymic organ development, EVA belonging to a novel immunoglobulin superfamily and its gene have been reported (Non-patent Document 4). However, there are no patent documents relating to thymus organ development factors and their use, and only thymic epithelial progenitor cells and techniques relating to their use are known (Patent Documents 1 and 2).
WO02 / 051988. WO03 / 105874. Nature, 372 (6501), 103-107, 1994. Developmental Biology, 195 (1), 195 (1), 1-15, 1998. Journal of Immunology, 164 (11), 5753-5760, 2000. Journal of Cell Biology, 141 (4), 1061-1071, 1998.

上述した通り、胸腺は健康維持にとって非常に重要な器官である。従って、胸腺器官発生の分子機構に係る全体像の解明とこれより得られる知見の活用は、人類の現在及び未来における生体防御、免疫システム、ホメオスタシス等の確保に極めて有力な手段となり得る。しかしながら、この分野の解明は未だ、遅々として進んでいない。 As mentioned above, the thymus is a very important organ for maintaining health. Therefore, the elucidation of the whole picture concerning the molecular mechanism of thymic organ development and the utilization of the knowledge obtained from this can be an extremely effective means for ensuring the defense of the human body, the immune system, homeostasis, etc. in the present and future. However, the elucidation of this field has not yet progressed.

本発明者らは、胸腺器官発生の分子機構に係る全体像の解明の重要性と必要性とを評価かつ認識し、この発生過程を徹底的に解明するための材料と方法につき、長年にわたる試行錯誤と創意工夫を重ねた結果、マウス胸腺原基で特異的に発現される遺伝子に着目し、その網羅的解析を行った。その成果として、胸腺器官発生因子ＴＯＦ１及びＴＯＦ２とこれ等の遺伝子を発見するに至った。その概要は次の通りある：
「先ず、胎齢11.5日のマウス胸腺原基をマイクロダイセクション法により単離し、対照表皮組織との差分ＰＣＲクローニング法により胸腺原基特異的遺伝子の候補クローンを得、それらの部分配列を網羅的に決定した。複数の出発材料に共通の発現が確認された遺伝子を多数同定した中に、Ｅｎｔｒｅｚ／ＮＣＢＩ公開遺伝子データベースにて性状解析が確認されずＥＳＴ名称のみ付与されている、新規な２つの遺伝子を発見した。これ等の遺伝子は胎齢11.5日のマウスでは胸腺器官ストロマ細胞のみに発現特異性がみられ、成体マウスでも胸腺での発現に比較的特異性が高いことを確認し、胸腺器官発生因子ＴＯＦ１（ｔｈｙｍｕｓ ｏｒｇａｎｏｇｅｎｅｓｉｓ ｆａｃｔｏｒ １）及びＴＯＦ２（ｔｈｙｍｕｓ ｏｒｇａｎｏｇｅｎｅｓｉｓ ｆａｃｔｏｒ ２）とそれぞれ命名した。」 The present inventors have evaluated and recognized the importance and necessity of elucidating the overall picture of the molecular mechanism of thymic organ development, and have made many years of trials on materials and methods for thoroughly elucidating this developmental process. As a result of repeated mistakes and ingenuity, we focused on genes that are specifically expressed in the mouse thymus primordia and conducted a comprehensive analysis. As a result, they have discovered thymus organ development factors TOF1 and TOF2 and their genes. The outline is as follows:
“First, the mouse thymus primordium at 11.5 gestational age was isolated by microdissection method, and candidate clones of thymus primordium-specific genes were obtained by differential PCR cloning with control epidermal tissue, and their partial sequences were comprehensively Among the many genes that have been confirmed to have common expression in multiple starting materials, two new genes that have not been confirmed in the Entrez / NCBI public gene database and that only the EST name has been assigned These genes were found to have specific expression only in thymic stromal cells in 11.5 day-old mice, and in adult mice, it was confirmed that their expression was relatively high in thymic organs. Factors TOF1 (thymus organogenesis factor 1) and TOF2 (thymus organogenesis f) act 2) "

ＴＯＦ１及びＴＯＦ２は、マウスの胸腺器官の発生過程において特異的に発現される新規な遺伝子である。従って、この事実は、これ等の遺伝子が胸腺器官発生、特に、胸腺器官および免疫系の形成に極めて重要な役割を演じ得ることを示唆する。更に、これ等のマウス遺伝子に対するホモロジー検索により得られるヒトの相同遺伝子は、胸腺発生異常、免疫不全、自己免疫疾患等の原因遺伝子あるいは責任遺伝子として、免疫病の診断、治療及び予防のための新規な手段をもたらし得る。その対象疾患として、例えば、ディジョージ症候群等の無胸腺症を含む先天性免疫不全症や、ＡＩＤＳや環境汚染等による後天的免疫不全症等々の免疫不全症を上げることができる。また、抗ガン剤使用の癌患者や造血細胞移植を含む臓器移植等に伴う免疫抑制状態の患者の治療にも適用できる。その具体的な治療方法として、例えば、ＴＯＦ１及び／又はＴＯＦ２並びこれ等の遺伝子を有効成分として用いる薬剤治療、遺伝子治療、細胞再生治療を上げることができる。また、これ等の遺伝子の定性検査や発現量の定量は、先天性及び後天性の免疫不全が疑わしい患者、更に、癌や、造血細胞移植等を含む臓器移植等に伴う免疫抑制状態の患者を対象にした免疫機能の診断あるいは臨床検査に用いることができる。   TOF1 and TOF2 are novel genes that are specifically expressed during the development of mouse thymic organs. This fact therefore suggests that these genes may play a vital role in thymic organ development, particularly in the formation of the thymic organ and immune system. Furthermore, human homologous genes obtained by homology search for these mouse genes are novel genes for diagnosis, treatment and prevention of immune diseases as causative genes or responsible genes for abnormal thymic development, immunodeficiency, autoimmune diseases, etc. Can provide a simple means. As the target disease, for example, congenital immunodeficiencies including athymosis such as DiGeorge syndrome, and acquired immunodeficiencies such as AIDS and environmental pollution can be raised. It can also be applied to the treatment of cancer patients using anticancer agents and immunosuppressed patients associated with organ transplantation including hematopoietic cell transplantation. Specific treatment methods include, for example, drug therapy, gene therapy, and cell regeneration therapy using TOF1 and / or TOF2 and these genes as active ingredients. In addition, qualitative examination of these genes and quantification of the expression level should be performed for patients with congenital and acquired immunodeficiency, as well as patients with immunosuppression due to cancer or organ transplantation including hematopoietic cell transplantation, etc. It can be used for diagnosis or clinical examination of the targeted immune function.

この発明によれば、上記課題を解決するための手段として、ＴＯＦ１及びＴＯＦ２両遺伝子ＤＮＡ、これ等の遺伝子ＤＮＡがコードするタンパク質ないしはポリペプチドあるいはペプチド、ハイブリダイゼーション用プローブ、及びＰＣＲ用プライマー等々、具体的には、次の（１）〜（９）に記載の物質がそれぞれ提供される：
配列番号１に記載の塩基配列は、ＴＯＦ１遺伝子ＤＮＡの塩基配列である。配列番
号１に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。更に、これ等のＤＮＡ分子の部分からなるＤＮＡ断片。
（２）配列番号２に記載の配列は、配列番号１に記載の完全長塩基配列をＲＳＡ１消化し得られた３´側のコード配列を占める合計３８０塩基対から作製された。配列番号２に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。更に、これ等のＤＮＡ分子の部分からなるＤＮＡ断片。
（３）配列番号３に記載の塩基配列は、ＴＯＦ２遺伝子ＤＮＡの完全長塩基配列である。
配列番号３に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。更に、これ等のＤＮＡ分子の部分からなるＤＮＡ断片。尚、配列番号３と後述の配列番号５に記載の両塩基配列はいずれも、ＴＯＦ２遺伝子ＤＮＡの完全長塩基配列であり、両者は互いにスプライシングバリアント（ｓｐｌｉｃｉｎｇ ｖａｒｉａｎｔ）ないしはａｌｔｅｒｎａｔｉｖｅ ｓｐｌｉｃｉｎｇ
ｆｏｒｍの関係にある。
（４）配列番号４に記載のアミノ酸配列は、ＴＯＦ２の完全長アミノ酸配列である。配列番号４に記載のアミノ酸配列、又は該アミノ酸配列において少なくとも１アミノ酸残基が置換又は欠失又は挿入されたアミノ酸配列を有するタンパク質。更に、これ等のアミノ酸配列の部分断片からなるタンパク質。尚、この明細書でいう置換とは別の種類のアミノ酸残基との置換を意味する。また、これ等のタンパク質は、胸腺器官発生に係る活性ドメインをその分子内に保有するか、胸腺器官発生活性を呈する必要がある。
（５）配列番号５に記載の塩基配列は、ＴＯＦ２遺伝子ＤＮＡの完全長塩基配列である。
配列番号５に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。更に、これ等のＤＮＡ分子の部分からなるＤＮＡ断片。
（６）配列番号６に記載のアミノ酸配列は、ＴＯＦ２の完全長アミノ酸配列である。配列番号６に記載のアミノ酸配列、又は該アミノ酸配列において少なくとも１アミノ酸残基が置換又は欠失又は挿入されたアミノ酸配列を有するタンパク質。更に、これ等のアミノ酸配列の部分断片からなるタンパク質。但し、これ等のタンパク質は、胸腺器官発生に係る
活性ドメインをその分子内に保有するか、胸腺器官発生活性を呈する必要がある。
（７）配列番号７に記載の配列は、配列番号３に記載の完全長塩基配列をＲＳＡ１消化し得られた５´側のコード配列を占める合計２４５塩基対から作製された。配列番号７に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。更に、これ等のＤＮＡ分子の部分からかるＤＮＡ断片。
（８）次に示す合計１５種のＴＯＦ１遺伝子用のＰＣＲプライマー。１種、又は２種（１対）を用いる：
ＴＯＦ１−１：５’−ＴＣＡＣＧＴＧＴＧＣＧＣＴＧＴＡＴＡＣＣ−３’
ＴＯＦ１−２：５’−ＣＣＴＴＴＧＣＣＴＣＴＣＣＴＧＣＡＣＴＴ−３’
ＴＯＦ１−３：５’−ＴＣＡＣＧＴＧＴＧＣＧＣＴＧＴＡＴＡＣＣ−３’
ＴＯＦ１−４：５’−ＡＡＧＣＴＧＧＡＣＡＡＡＣＴＡＡＧＧＡＣ−３’
ＴＯＦ１−５：５’−ＡＴＡＣＡＧＣＧＣＡＣＡＣＧＴＧＡＧＴＧ−３’
ＴＯＦ１−６：５’−ＴＴＣＣＴＡＴＴＴＧＧＣＣＡＧＣＡＡＣＣ−３’
ＴＯＦ１−７：５’−ＴＡＣＣＣＡＣＴＧＧＴＣＡＣＣＡＣＡＡＴＧ−３’
ＴＯＦ１−８：５’−ＡＣＡＴＣＴＣＴＣＣＡＴＣＣＡＧＴＴＣＣ−３’
ＴＯＦ１−９：５’−ＣＣＣＴＴＴＧＣＣＴＣＴＣＣＴＧＣＡＣＴＴＣＣＡＣ−３’
ＴＯＦ１−１０：５’−ＡＧＣＧＧＧＴＡＴＡＣＡＧＣＧＣＡＣＡＣＧＴＧＡＧ−３’
ＴＯＦ１−１１：５’−ＧＣＧＡＴＧＧＡＧＧＡＴＧＧＡＴＧＣＴＣＴＣＣＧＧ−３’
ＴＯＦ１−１２：５’−ＡＧＣＣＴＧＴＧＴＧＧＡＣＣＴＧＧＧＣＧＣＣＴＡＧ−３’
ＴＯＦ１−１３：５’−ＣＴＡＧＧＣＧＣＣＣＡＧＧＴＣＣＡＣＡＣＡＧＧＣＴ−３’
ＴＯＦ１−１４：５’−ＧＡＧＧＧＴＴＴＣＣＴＧＧＴＣＣＣＴＧＧＧＡＴＧＧＧＣ
Ｃ−３’
ＴＯＦ１−１５：５’−ＧＧＣＣＣＣＡＧＴＧＴＧＧＴＣＧＣＡＣＴＧＡＧＡＧＴＴ
Ｇ−３’
（９）次に示す合計６種のＴＯＦ２遺伝子用のＰＣＲプライマー。例えば、ＴＯＦ２−１ＦとＴＯＦ２−１Ｒとの組合せ（１対）を用いる：
ＴＯＦ２−１Ｆ：５’−ＡＧＴＴＧＣＡＧＴＣＣＴＴＧＴＴＣＴＧＧ−３’
ＴＯＦ２−１Ｒ：５’−ＣＴＧＴＣＴＣＣＣＡＡＡＣＴＧＧＧＴＡＴＣ−３’
ＴＯＦ２−２Ｆ：５’−ＡＴＧＡＡＧＧＴＧＡＴＣＴＴＣＴＣＡＧＴＴＧ−３’
ＴＯＦ２−２Ｒ：５’−ＴＣＡＧＴＡＡＣＴＴＴＴＡＴＣＡＧＴＧ−３’
ＴＯＦ２−３Ｆ：５’−ＧＴＧＴＧＧＡＣＴＴＣＣＣＴＴＧＣＡＧＴ−３’
ＴＯＦ２−３Ｒ：５’−ＴＧＣＴＧＣＣＡＣＡＴＡＴＡＧＧＣＴＣＡ−３ According to the present invention, as means for solving the above problems, both TOF1 and TOF2 gene DNAs, proteins or polypeptides or peptides encoded by these gene DNAs, hybridization probes, PCR primers, etc. Specifically, the following substances (1) to (9) are provided:
The base sequence described in SEQ ID NO: 1 is the base sequence of TOF1 gene DNA. A DNA molecule having the base sequence set forth in SEQ ID NO: 1 or a base sequence in which at least one base in the base sequence is substituted based on codon degeneracy. Furthermore, a DNA fragment consisting of a part of these DNA molecules.
(2) The sequence described in SEQ ID NO: 2 was prepared from a total of 380 base pairs occupying the 3 ′ coding sequence obtained by digesting the full-length base sequence described in SEQ ID NO: 1 with RSA1. A DNA molecule having the base sequence set forth in SEQ ID NO: 2 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons. Furthermore, a DNA fragment consisting of a part of these DNA molecules.
(3) The base sequence described in SEQ ID NO: 3 is the full-length base sequence of TOF2 gene DNA.
A DNA molecule having the base sequence set forth in SEQ ID NO: 3 or a base sequence in which at least one base in the base sequence is substituted based on codon degeneracy. Furthermore, a DNA fragment consisting of a part of these DNA molecules. Both of the nucleotide sequences described in SEQ ID NO: 3 and SEQ ID NO: 5 described later are full-length nucleotide sequences of TOF2 gene DNA, and both are splicing variants or alternative splicing to each other.
There is a form relationship.
(4) The amino acid sequence set forth in SEQ ID NO: 4 is the full-length amino acid sequence of TOF2. A protein having the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence in which at least one amino acid residue is substituted, deleted or inserted in the amino acid sequence. Furthermore, proteins consisting of partial fragments of these amino acid sequences. In addition, the substitution referred to in this specification means substitution with an amino acid residue of a different type. In addition, these proteins must have an active domain involved in thymus organ development in their molecules or exhibit thymus organ development activity.
(5) The base sequence described in SEQ ID NO: 5 is the full-length base sequence of TOF2 gene DNA.
A DNA molecule having the base sequence set forth in SEQ ID NO: 5 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons. Furthermore, a DNA fragment consisting of a part of these DNA molecules.
(6) The amino acid sequence set forth in SEQ ID NO: 6 is the full-length amino acid sequence of TOF2. A protein having the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence in which at least one amino acid residue is substituted, deleted, or inserted in the amino acid sequence. Furthermore, proteins consisting of partial fragments of these amino acid sequences. However, these proteins must have an active domain related to thymus organ development in the molecule or exhibit thymus organ development activity.
(7) The sequence described in SEQ ID NO: 7 was prepared from a total of 245 base pairs occupying the 5 ′ coding sequence obtained by digesting the full-length base sequence described in SEQ ID NO: 3 with RSA1. A DNA molecule having the base sequence set forth in SEQ ID NO: 7 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons. In addition, DNA fragments made from portions of these DNA molecules.
(8) PCR primers for a total of 15 TOF1 genes shown below. Use one or two (one pair):
TOF1-1: 5′-TCACGTGTGCCGCTGTATACC-3 ′
TOF1-2: 5′-CCTTTGCCTCTCTCGCACTTT-3 ′
TOF1-3: 5′-TCACGTGTGCCGCTGTATACC-3 ′
TOF1-4: 5′-AAGCTGGGACAAACTAGAGAC-3 ′
TOF1-5: 5′-ATACACGGCACACGTGGAGTG-3 ′
TOF1-6: 5′-TTCCCTATTGGCCAGCAACC-3 ′
TOF1-7: 5′-TACCCACTGGTCACCACAATG-3 ′
TOF1-8: 5′-ACATCTTCTCCATCCAGTTCCC-3 ′
TOF1-9: 5′-CCCTTTGCCCTCTCCTGCACTTCCC-3 ′
TOF1-10: 5′-AGCGGGTATACAGCGCACACGTGAG-3 ′
TOF1-11: 5′-GCGATGGAGGGATGGATGCTCTCCGG-3 ′
TOF1-12: 5′-AGCCTGTGTGACCCTGGGCGCTAG-3 ′
TOF1-13: 5′-CTAGGCGCCCAGGTCCACACAGGGCT-3 ′
TOF1-14: 5′-GAGGGTTTCCTGGTCCCTGGGATGGGGC
C-3 '
TOF1-15: 5'-GGCCCCAGTGTGGTCGCACTGAGAGTTT
G-3 '
(9) PCR primers for a total of 6 types of TOF2 genes shown below. For example, a combination (one pair) of TOF2-1F and TOF2-1R is used:
TOF2-1F: 5′-AGTTGCAGTCCCTTGTTCTG-3 ′
TOF2-1R: 5′-CTGTCTCCCAAACTGGGTATC-3 ′
TOF2-2F: 5′-ATGAAGGTGATCTTTCCAGTTG-3 ′
TOF2-2R: 5′-TCAGTAACTTTTATCAGTG-3 ′
TOF2-3F: 5'-GTTGTGACTACTCCCTTGCAGT-3 '
TOF2-3R: 5'-TGCTGCCCACATAGGCTCA-3

以下、参考例及び実施例を上げ、本発明の構成と効果を具体的に説明する。但し、この発明は、これ等の参考例や実施例だけに限定されるわけではない。
（参考例１） The configuration and effects of the present invention will be specifically described below with reference examples and examples. However, the present invention is not limited only to these reference examples and examples.
(Reference Example 1)

胎齢１１．５日胚におけるマウス胸腺原基の同定：
胎齢１１．５日胚のＣ５７ＢＬ／６Ｊ系統マウスから胸腺原基を含む組織切片を作製した。正確に胸腺原基を構築する上皮系細胞領域を確定するために、抗マウスＣＤ４５及び汎サイトケラチン抗体（ｅ−Ｂｉｏｓｃｉｅｎｃｅ社［米国］製）を用いて、胸腺原基を含む選定した組織切片を二重染色し、胸腺原基とその周囲に集積するＴ前駆細胞を含む血球細胞を同定した。
（参考例２） Identification of mouse thymic primordium in embryonic day 11.5 embryos:
Tissue sections containing thymic primordia were prepared from C57BL / 6J strain mice with embryonic day 11.5 days. In order to determine the epithelial cell region that accurately constructs the thymic primordia, anti-mouse CD45 and pancytokeratin antibody (manufactured by e-Bioscience [USA]) were used to select selected tissue sections containing thymic primordia. Double staining was performed to identify blood cells containing thymic primordia and T progenitor cells that accumulate around them.
(Reference Example 2)

マイクロダイセクション法を利用した胸腺原基由来RNAの単離：
組織切片から正確に胸腺原器のみを単離するために、マイクロダイセクション法を利用した（ＰｉｘＣｅｌｌIIＬａｓｅｒ Ｃａｐｔｕｒｅ Ｍｉｃｒｏｄｉｓｓｅｃｔｉｏｎシステム；Ａｒｃｔｕｒｕｓ社［米国］製）。得られた組織からのｔｏｔａｌ ＲＮＡ抽出は、ＰｉｃｏＰｕｒｅ ＲＮＡ Ｉｓｏｌａｔｉｏｎ Ｋｉｔ（Ａｒｃｔｕｒｕｓ社［米国］製）を使用した。
（参考例３） Isolation of RNA from thymic primordium using microdissection method:
A microdissection method was used (PixCellII Laser Capture Microdetection System; manufactured by Arcturus [USA]) to accurately isolate only the thymus progenitor from the tissue section. For the total RNA extraction from the obtained tissue, PicoPure RNA Isolation Kit (manufactured by Arcturus [USA]) was used.
(Reference Example 3)

差分ＰＣＲクローニング法による胸腺器官関連遺伝子の同定：
Ｔｏｔａｌ ＲＮＡは、６胎仔から単離された胸腺原基から抽出され、そのｃＤＮＡへの変換及び増幅は、Ｓｕｐｅｒ ＳＭＡＲＴ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社［米国］製）を用いて行われた。得られた胸腺原基由来のｃＤＮＡと、同様の操作により得られた同齢表皮組織ｃＤＮＡを利用してＰＣＲ−ｓｅｌｅｃｔｅｄｃＤＮＡ ｓｕｂｔｒａｃｔｉｏｎ Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社［米国］製）による差分ＰＣＲクローニングを行った。差分ｃＤＮＡ産物はｐＧＥＭ−Ｔｅａｓｙベクターにクローニングされた。１回のＰＣＲクローニングで２００−２５０クローンを得る作業を５回繰り返し、合計１３７０クローンを得、その核酸配列を決定した。それぞれのロットから得られた核酸配列をＥｎｔｒｅｚ／ＮＣＢＩゲノム・データベースにて解析し推定される遺伝子群を同定した。これらの遺伝子群の中ですべてのロットに含まれていた遺伝子群を胸腺原基に特異的に発現する候補遺伝子として選定した。
（参考例４） Identification of thymus organ-related genes by differential PCR cloning:
Total RNA was extracted from thymic primordia isolated from 6 fetuses, and the conversion and amplification into cDNA were performed using Super SMART cDNA Synthesis Kit (Clontech [USA]). Differential PCR cloning by PCR-selected cDNA subtraction kit (Clontech [USA]) was performed using the obtained thymic primordium-derived cDNA and the same-aged epidermal tissue cDNA obtained by the same operation. The differential cDNA product was cloned into the pGEM-Teasy vector. The operation of obtaining 200-250 clones by one PCR cloning was repeated 5 times to obtain a total of 1370 clones, and their nucleic acid sequences were determined. Nucleic acid sequences obtained from each lot were analyzed in the Entrez / NCBI genome database to identify a presumed gene group. Among these gene groups, the gene groups contained in all lots were selected as candidate genes that are specifically expressed in the thymus primordia.
(Reference Example 4)

候補遺伝子群の発現解析：
候補遺伝子群の発現解析は、定量的ＰＣＲ法が利用され、胎齢１１．５日、１４．５日及び生後８週齢マウスの各臓器別あるいは細胞系譜別にその発現量が比較された。胎齢１１．５日の各組織を単離するためにマイクロダイセクション法を利用した。これら組織から得られたＲＮＡは、ＲｉｂｏＡｍｐ ＲＮＡ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ａｒｃｔｕｒｕｓ社［米国］製）でＲＮＡ増幅され、更にＳｕｐｅｒ ＳｃｒｉｐｔII逆転写酵素（インビトロジェン社）でｃＤＮＡに変換された。定量発現解析には、リアルタイムＰＣＲ法（ＡＢＩ Ｐｒｉｓｍ ７９００ＨＴ型；ＡＢＩ社［米国］製）が利用された。
（参考例５） Expression analysis of candidate genes:
For the expression analysis of candidate gene groups, the quantitative PCR method was used, and the expression levels were compared for each organ or cell lineage of embryonic day 11.5 day, 14.5 day and 8 week old mice. A microdissection method was used to isolate each tissue at 11.5 days of gestational age. RNA obtained from these tissues was amplified with RiboAmp RNA Amplification Kit (manufactured by Arcturus [USA]) and further converted to cDNA with Super Script II reverse transcriptase (Invitrogen). For quantitative expression analysis, a real-time PCR method (ABI Prism 7900HT type; manufactured by ABI [USA]) was used.
(Reference Example 5)

Ｗｈｏｌｅ−ｍｏｕｎｔ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ：
このｈｙｂｒｉｄｉｚａｔｉｏｎ法は、Ｍａｎｌｅｙらの方法に準じて行うことができる（Ｄｅｖｅｌｏｐｍｅｎｔ，１２１，１９８９−２００３，１９９５文献１）。４容量％パラホルムアルデヒドで固定した胎齢９−１２日胚をメタノールで脱水後、ジゴキシンで標識したＴＯＦ１及びＴＯＦ２のＲＮＡプローブ（塩基配列２及び７に記載）を用いてハイブリダイゼーションを行う。ＴＯＦ１のＲＮＡプローブは、ＲＳＡＩ消化により得られるＴＯＦ１の３´側のコード配列（３８０塩基対）を用いることができる。また、ＴＯＦ２のものは、５´側翻訳開始部位から２４５塩基対のコード配列を利用することができる。 Whole-mount in situ hybridization:
This hybridization method can be performed according to the method of Manley et al. (Development, 121, 1989-2003, 1995, literature 1). Embryonic day 9-12 day embryos fixed with 4% by volume paraformaldehyde are dehydrated with methanol, and then hybridized using digoxin-labeled TOF1 and TOF2 RNA probes (described in nucleotide sequences 2 and 7). As the TOF1 RNA probe, the coding sequence (380 base pairs) on the 3 ′ side of TOF1 obtained by RSAI digestion can be used. The TOF2 can use a coding sequence of 245 base pairs from the 5 ′ translation start site.

候補遺伝子群の発現解析：
候補遺伝子群の発現解析は、定量的ＰＣＲ法が利用され、胎齢１１．５日、１４．５日及び生後８週齢マウスの各臓器別あるいは細胞系譜別にその発現量が比較された。胎齢１１．５日の各組織を単離するためにマイクロダイセクション法を利用した。これら組織から得られたＲＮＡは、ＲｉｂｏＡｍｐ ＲＮＡ Ａｍｐｌｉｆｉｃａｔｉｏｎ Ｋｉｔ（Ａｒｃｔｕｒｕｓ社［米国］製）でＲＮＡ増幅され、更にＳｕｐｅｒ ＳｃｒｉｐｔII逆転写酵素（インビトロジェン社）でｃＤＮＡに変換された。定量発現解析には、リアルタイムＰＣＲ法（ＡＢＩ Ｐｒｉｓｍ ７９００ＨＴ型；ＡＢＩ社［米国］製）が利用された
候補遺伝子群のうち、ＴＯＦ１の結果を図１に、ＴＯＦ２のそれを図２にそれぞれ示す。これ等の両遺伝子はいずれも、胎齢11.5日のマウスでは胸腺器官ストロマ細胞のみに発現特異性がみられ、また、成体マウスでも胸腺での発現に比較的特異性の高いことが確認された。 Expression analysis of candidate genes:
For the expression analysis of candidate gene groups, the quantitative PCR method was used, and the expression levels were compared for each organ or cell lineage of embryonic day 11.5 day, 14.5 day and 8 week old mice. A microdissection method was used to isolate each tissue at 11.5 days of gestational age. RNA obtained from these tissues was amplified with RiboAmp RNA Amplification Kit (manufactured by Arcturus [USA]) and further converted to cDNA with Super Script II reverse transcriptase (Invitrogen). Among the candidate gene groups for which the real-time PCR method (ABI Prism 7900HT type; manufactured by ABI [USA]) is used for quantitative expression analysis, the results of TOF1 are shown in FIG. 1, and those of TOF2 are shown in FIG. Both of these genes were found to have expression specificity only in thymic stromal cells in embryonic day 11.5 mice, and in adult mice it was confirmed that they were relatively specific for expression in the thymus.

本発明に係るＴＯＦ１及び２とこれ等の遺伝子は、胸腺不全あるいは免疫不全に起因する種々の疾患の基礎研究試薬、治療剤、治療薬の開発、診断剤、遺伝子治療、遺伝子診断、予防等に利用できる。 TOF1 and 2 and their genes according to the present invention are used for basic research reagents, therapeutic agents, development of therapeutic agents, diagnostic agents, gene therapy, genetic diagnosis, prevention, etc. for various diseases caused by thymic or immunodeficiency Available.

器官別（横軸）にＴＯＦ１遺伝子の発現量（縦軸）を示す図である。（ａ）胎齢１１．５日、（ｂ）胎齢１４．５日、及び（ｃ）生後８週齢の各マウス。It is a figure which shows the expression level (vertical axis) of TOF1 gene according to organ (horizontal axis). (A) 11.5 days of gestational age, (b) 14.5 days of gestational age, and (c) 8 weeks old mice. 器官別（横軸）にＴＯＦ２遺伝子の発現量（縦軸）を示す図である。（ａ）胎齢１１．５日、（ｂ）胎齢１４．５日、及び（ｃ）生後８週齢の各マウス。It is a figure which shows the expression level (vertical axis) of TOF2 gene according to organ (horizontal axis). (A) 11.5 days of gestational age, (b) 14.5 days of gestational age, and (c) 8 weeks old mice.

Claims (7)

配列番号１に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。   A DNA molecule having the base sequence set forth in SEQ ID NO: 1 or a base sequence in which at least one base in the base sequence is substituted based on codon degeneracy. 配列番号２に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。   A DNA molecule having the base sequence set forth in SEQ ID NO: 2 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons. 配列番号３に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。   A DNA molecule having the base sequence set forth in SEQ ID NO: 3 or a base sequence in which at least one base in the base sequence is substituted based on codon degeneracy. 配列番号４に記載のアミノ酸配列、又は該アミノ酸配列において少なくとも１アミノ酸残基が置換又は欠失又は挿入されたアミノ酸配列を有するタンパク質。   A protein having the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence in which at least one amino acid residue is substituted, deleted or inserted in the amino acid sequence. 配列番号５に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。 A DNA molecule having the base sequence set forth in SEQ ID NO: 5 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons. 配列番号６に記載のアミノ酸配列、又は該アミノ酸配列において少なくとも１アミノ酸残基が置換又は欠失又は挿入されたアミノ酸配列を有するタンパク質。 A protein having the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence in which at least one amino acid residue is substituted, deleted, or inserted in the amino acid sequence. 配列番号７に記載の塩基配列、又は該塩基配列において少なくとも１塩基がコドンの縮重に基づき置換された塩基配列を有するＤＮＡ分子。 A DNA molecule having the base sequence set forth in SEQ ID NO: 7 or a base sequence in which at least one base in the base sequence is substituted based on degeneracy of codons.
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