㧏វᗐ TSA-FISH Ἢࢅ⏕࠷ࡒཋᰶ⏍∸ࡡᶭ⬗㐿ఎᏄࡡ᳠ฝỀᅥᅰቫ⎌ሾโᚒ◂✪ᐄࠈᕖ୕࿔ྒྷ
ᣞᑙᩅဤࠈኬᶣᬏⰃ
1. ◂✪⫴ᬊ࡛┘Ⓩ
ࠈFluorescence in-situ hybridization (FISH) ἪࡢࠉᙟឺⓏ࡞ㆉื࠿ᅏ㞬࡝⎌ሾ୯ࡡ᚜⏍∸⩄࡞ᑊࡊࠉ≁ᏽࡡ᚜⏍∸⩄ࡡࡲࢅ in-situ ࠾ࡗࢨࣤࢡࣜࢬ࡚ࣜࣝ࣊ࣜ᳠ฝࡌࡾࡆ࡛࠿ྊ⬗࡚࠵ࡾࠊ୯࡚ࡵ rRNA ࢅᵾⓏ࡛ࡊࡒ FISHἪࡢࠉrRNA 㐿ఎᏄ㒼า࡞ᇱࡘ࠷ࡒ⣌⤣ฦ㢦Ꮥ୕࡚ࡡྜྷᏽ࠿ྊ⬗࡝ࡆ࡛࠾ࡼࠉ⎌ሾ᚜⏍∸ᕝᏕࡡฦ㔕࡚ࡵᗀࡂ⏕࠷ࡼࡿ࡙࠷ࡾࠊ㎾ᖳ rRNA ࢅᵾⓏ࡛ࡊࡒ FISH Ἢࡡ௙࡞ࠉ᚜⏍∸ࡡ⏍⌦ᏕⓏὩᛮࡷ⏍⌦ᏕⓏᶭ⬗࣎ࢷࣤࢨࣔࣜࢅフ౮࡚ࡀࡾ mRNA ࡷ᯹Ⰵమ୕࡞ࢤ࣭ࢺࡈࡿࡒᶭ⬗㐿ఎᏄࢅᵾⓏ࡛ࡊࡒ FISH Ἢ࠿ሒ࿈ࡈࡿጙࡴ࡙࠷
ࡾࠊࡆࡿࡱ࡚࡞ mRNA ࢅ᳠ฝࡊࡒౚࡢ 8 ሒࠉᶭ⬗㐿ఎᏄࢅ᳠ฝࡊࡒౚࡢ 1 ሒ࠵ࡾ࠿ࠉ⎌ሾ᚜⏍∸࡞㐲⏕ࡌࡾ࡞ࡢㄚ㢗࠿ṟࡈࡿ࡙࠷ࡾࠊ୯࡚ࡵ᳠ฝវᗐࡡ୘㊂ࡢࠉࢤࣅ࣭ᩐࡡᑛ࡝࠷ mRNA ࡷᶭ⬗㐿ఎᏄࢅ᳠ฝࡌࡾ
㝷ࡡኬࡀ࡝ၡ㢗࡚࠵ࡾࠊࡐࡡࡒࡴ mRNA ࡷᶭ⬗㐿ఎᏄࢅ᳠ฝࡊࡒኣࡂࡡሒ࿈࡞࠽࠷࡙ࠉࡻࡽ㧏វᗐ᳠ฝ࠿
ྊ⬗࡝ᩐⓊሲᇱ࠾ࡼ࡝ࡾ࣎ࣛࢽࢠ࢛ࣝࢲࢺࣈ࣭ࣞࣇ (࣎ࣛࣈ࣭ࣞࣇ) ࠿⏕࠷ࡼࡿ࡙࠽ࡽࠉ௑᪝ࡱ࡚࡞။ୌࠉᶭ⬗㐿ఎᏄࢅ᳠ฝࡊࡒሒ࿈࡞࠽࠷࡙ࡵ࣎ࣛࣈ࣭ࣞࣇ࠿⏕࠷ࡼࡿ࡙࠷ࡾࠊୌ᪁ࠉ18~25 ሲᇱ࠾ࡼ࡝ࡾ࢛ࣛࢥࢽ
ࢠ࢛ࣝࢲࢺࣈ࣭ࣞࣇ (࢛ࣛࢥࣈ࣭ࣞࣇ) ࡢࠉ࣎ࣛࣈ࣭ࣞࣇ࡞Ẓ࡬࣐ࢪ࣏ࢴࢲㆉื⬗ງ࠿㧏ࡂࠉࣈ࣭ࣞࣇスゝࡡ⮤⏜ᗐ࠿㧏࠷࡞ࡵ㛭ࢂࡼࡍࠉវᗐࡡၡ㢗࠾ࡼ mRNA ࡷᶭ⬗㐿ఎᏄࡡ᳠ฝ࡞ࡢ࠵ࡱࡽ⏕࠷ࡼࡿ࡙࠷࡝࠷ࡡ࠿⌟≟࡚࠵ࡾࠊࠈᠻࠍࡡࢡ࣭ࣜࣈࡢ two-pass tyramide signal amplification (TSA)-FISH Ἢࢅ⏕࠷ࡾࡆ࡛࡚ࠉ࢛ࣛࢥࣈ࣭ࣞࣇ࡚
ࡵ mRNA ࢅಘ㢏ᛮࡡ࠵ࡾࢨࢡࢻ࡚ࣜ᳠ฝ࡚ࡀࡾࡆ࡛ࢅሒ࿈ࡊࡒࠊTwo-pass TSA-FISH Ἢࡢࠉhorseradish
peroxidase (HRP) ࡡ㓕⣪り፳ཬᚺ࡞ࡻࡾࢨࢡࢻࣜቌᖕࢅ⧖ࡽ㏁ࡌࡆ࡛࡚ࠉTSA-FISH Ἢ࡞Ẓ࡬ⴥࡊࡂ㧏࠷᳠ฝវᗐࢅᚋࡾࡆ࡛࠿࡚ࡀࡾ (TSA-FISH Ἢࡢ㏳ᖏࡡ FISH Ἢࡡ⣑ 20 ಶࡡ㍜ᗐ࠿ᚋࡼࡿࡾࡡ࡞ᑊࡊࠉtwo-passTSA-FISH Ἢࡢ⣑ 400 ಶ࡛ࡡモ⟤࠿࠵ࡾ)ࠊࡐࡆ࡚ᠻࠍࡢࡆࡡ two-pass TSA-FISH Ἢࢅ⏕࠷ࡿࡣࠉវᗐ࠿୘༎
ฦ࡝ࡒࡴᅏ㞬࡚࠵ࡖࡒ࢛ࣛࢥࣈ࣭ࣞࣇࢅ⏕࠷ࡒᶭ⬗㐿ఎᏄࡡ᳠ฝ࠿ྊ⬗࡝ࡡ࡚ࡢ࡝࠷࠾࡛⩻࠻ࡒࠊᮇ◂✪࡚
ࡢࠉtwo-pass TSA-FISH Ἢࢅ⏕࠷࡙ࠉ࢛ࣛࢥࣈ࣭ࣞࣇ࡚᚜⏍∸ࡡࢨࣤࢡࣜࢤࣅ࣭㐿ఎᏄ࠿᳠ฝྊ⬗࠾᳠ゞࡊࡒࠊ
2. ᐁ㥺᪁Ἢ

ࠈ

2.1 ࣓ࢸࣜ᚜⏍∸࡛ᵾⓏ㐿ఎᏄࠈ࣓ࢸࣜ᚜⏍∸࡞ࡢࠉධࢣࢿ࣑㒼า࠿よㄖࡈࡿࠉ⣵⬂ባฌ⌦ࢅ᪃ࡈ࡝ࡂ࡛ࡵ TSA-FISH Ἢ࠿㐲⏕ྊ⬗࡝
Methanococcus maripaludis S2 ᰬ (JCM13030) ࢅ⏕࠷ࡒࠊࡱࡒࠉࢾ࢝ࢷ࢔ࣇࢤࣤࢹ࣭࡛ࣞࣜࡊ࡙ Methanococcus
vanneilii (DSM1229) ࢅ⏕࠷ࡒࠊ୦⩽ࡢᑊᩐቌṢ᭿࡞ᅂ཭ࡊࠉࣂ࣑ࣚ࣌ࣜ࢓ࣜࢸࣃࢺ࡚ᅖᏽࡊࡒࠊࠈᵾⓏ㐿ఎᏄࡢࠉM.maripaludis S2 ᰬࡡࢣࢿ࣑୕࡞ࢨࣤࢡࣜࢤࣅ࣭࡛ࡊ࡙Ꮛᅹࡌࡾ Methyl coenzyme M
reductase (mcr) ࡛ࡊࡒࠊࣈ࣭ࣞࣇࡢࠉmcr ࡡ alpha subnuit ࡞≁␏Ⓩ࡝ࣈࣚ࢕࣏࣭࡛ࡊ࡙▩ࡼࡿࡾ ME1ࠉME2ࠉME3ࠉMR1 ࡡ 4 ⛸ࢅࡐࡿࡑࡿ M.maripaludis ࡡࢬࣤࢪ㙈࡞≁␏Ⓩ࡞スゝࡊࡒࠊࡱࡒࠉࣈ࣭ࣞࣇ࡛ᵾⓏฦᏄ࡛
ࡡば࿰ᛮࢅ㧏ࡴࡾࡒࡴ࡞ࣈ࣭ࣞࣇࡡୌ㒂ࢅ locked nucleic acid (LNA) ࡞⨠ᥦࡊࡒࠊࣈ࣭ࣞࣇࡡ୦❻࡞ࡢ DIG
ࢅಞ㣥ࡊࡒࠊ
2.2 TSA-FISH ἪࠈTSA-FISH Ἢࡢࠉ௧ୖࡡᡥ㡨࡚⾔ࡖࡒࠊࡱࡍࠉ᯹Ⰵమ࡛ࣈ࣭ࣞࣇࢅንᛮࡈࡎࡒᚃࠉྜྷ᫤࡞ 4 ⛸ࡡࣈ࣭ࣞࣇ
ࢅஹ㞟ࡈࡎࡒࠊࡐࡡᚃࠉᢘཋᢘమཬᚺ࡞ࡻࡽ anti-DIG-HRP ࢅ⤎ྙࡈࡎ Tyramide-Cy3 ࢅ⏕࠷ࡒ TSA ཬᚺ࡞ࡻࡽྊっ໩ࡊࡒࠊ
2.3 Two-pass TSA-FISH ἪࠈTwo-pass TSA-FISH Ἢࡢࠉ୕㏑ࡡ TSA-FISH Ἢ࡛ྜྷᵕ࡞ 4 ⛸ࡡࣈ࣭ࣞࣇࢅஹ㞟ࡈࡎࡒᚃࠉᢘཋᢘమཬᚺ࡞

ࡻࡽ anti-DIG-HRP ࢅ⤎ྙࡈࡎࠉTyramide-DNP ࢅ⏕࠷ࡒ TSA ཬᚺࢅ⾔࠷ࠉDNP ࢅⳞమහ࡞ỷ╌ࡈࡎࡒࠊ⤾࠷࡙ anti-DNP-HRP ࢅᢘཋᢘమཬᚺ࡞ࡻࡽ⤎ྙࡈࡎࠉTSA ཬᚺ࡞ࡻࡽ Tyramide-Cy3 ࢅỷ╌ࡈࡎࠉྊっ໩ࡊࡒࠊ
3. ⤎ᯕ࡛⩻ᐳ3.1 TSA-FISH Ἢࠉtwo-pass TSA-FISH Ἢ࡞ࡻࡾ᳠ฝ
ࠈࡱࡍ M.maripaludis ࡞ᑊࡊ࡙ TSA-FISH Ἢࢅ㐲⏕ࡊࡒ࡛ࡆࢀࠉⳞమ࠾ࡼࡢධࡂ⺧ක࠿ᚋࡼࡿ࡝࠾ࡖࡒ (Fig.1-Aࠉ㟚ක᫤㛣 : 1000ms)ࠊࡐࡆ࡚ࠉtwo-pass TSA-FISH Ἢࢅ㐲⏕ࡊࡒ࡛ࡆࢀࠉⳞమ࠾ࡼᙁ࠷⺧ක࠿ᚋࡼࡿࡒ (Fig.1-Bࠉ㟚ක᫤㛣 :100ms)ࠊࡊ࠾ࡊࠉࡌ࡬࡙ࡡⳞమ࠾ࡼ⺧කࢅᚋࡾࡆ࡛ࡢ࡚ࡀ
࡝࠾ࡖࡒࠊࡱࡒࠉ⺧කࡢⳞධమ࠾ࡼୌᵕ࡞ᚋࡼࡿࡾࡵࡡ࡚
ࡢ࡝ࡂࠉᑻᡜⓏ࡞ᚋࡼࡿࡒࠊࡆࡿࡢࠉⳞమහࡡᵾⓏ㐿ఎᏄࡡ㎾എ࡚ࢨࢡࢻࣜቌᖕ࠿⏍ࡋࡒࡒࡴࡓ࡛⩻࠻ࡼࡿࡾࠊ
ࠈḗ࡞ࠉᚋࡼࡿࡒ⺧ක࠿ᵾⓏ㐿ఎᏄ࡞ஹ㞟ࡊࡒࣈ࣭ࣞࣇ⏜ᮮ࡚࠵ࡾ࠾☔ヾࡌࡾࡒࡴ࡞ M.maripaludis ࡞ᑊࡊ DNase ฌ⌦ࢅ⾔࠷ࠉᵾⓏ㐿ఎᏄࢅᾐ໩ࡊࡒࠊࡐࡡ⤎ᯕࠉⳞమ࠾ࡼࡡ
ࢨࢡࢻࣜࡢධࡂふᐳࡈࡿ࡝ࡂ࡝ࡽࠉ⺧ක࠿ᵾⓏ㐿ఎᏄ࡞ஹ㞟ࡊࡒࣈ࣭ࣞࣇ⏜ᮮ࡚࠵ࡾࡆ࡛࠿☔ヾࡈࡿࡒࠊ
ࠈࡱࡒࠉFA ⃨ᗐ࡛ཬᚺῺᗐࢅ㧏ࡂࡌࡾࡆ࡛࡚ࣀ࢕ࣇࣛࢱ

Fig.1 Detection of mcrA gene in Methanococcus
maripaludis strain S2 cells by CARD-FISH (A) or by twopass CARD-FISH (B). Circles show unspecific signals.
DAPI (left) and epifluorescence micrograph (right) show
identical fields. Exposure time for right panel (A) was
1000ms, and right panel (B) was 100ms.

࢕ࢭ࣭ࢨࣘࣤ᫤࡞࠽ࡄࡾࢪࢹࣛࣤࢩ࢘ࣤࢨ࣭ࢅ୕ࡅࠉࣈ࣭ࣞࣇ࠿᯹Ⰵమ࠾ࡼ๡㞫ࡌࡾࡆ࡛࡞ࡻࡾࢨࢡࢻࣜࡡᾐකࢅ☔ヾࡊࡻ࠹࡛ࡊࡒ࡛ࡆࢀࠉ㧏ࢪࢹࣛࣤࢩ࢘ࣤࢨ࣭ୖ (eg.࣑࣌ࣜ࢓࣐ࢺ⃨ᗐ 90%ࠉཬᚺῺᗐ 48˚C) ࡚ࡵᏰධ࡞ࢨࢡࢻࣜࢅᾐࡌ஥ࡢ࡚ࡀ࡝࠾ࡖࡒࠊࡊ࠾ࡊࠉࢾ࢝ࢷ࢔ࣇࢤࣤࢹ࣭࡚ࣞࣜ࠵ࡾ M.vannielii (4 ⛸ࡡࣈ࣭ࣞࣇ࡛ࡡ࣐ࢪ࣏ࢴࢲࡢ 1~3 ሲᇱ) ࡞࠽࠷࡙ࡢࠉ఩ࢪࢹࣛࣤࢩ࢘ࣤࢨ᮪࣭௲ୖ࡚ぜࡼࡿࡒ㟸≁␏Ⓩ⺧ක࠿ࠉࢪࢹࣛࣤࢩ࢘ࣤࢨ࣭ࢅ୕ࡅ࡙࠷ࡂ஥࡚ぜࡼࡿ࡝ࡂ࡝ࡖࡒࠊࡆࡡࡆ࡛ࡢࠉ本技術が࣐ࢪ࣏ࢴࢲࢅㆉืࡌࡾ≁␏ᛮࢅ᭯ࡊ࡙࠷ࡾࡆ࡛ࢅ♟ြࡊ࡙࠷ࡾࠊ௑ᚃࠉ࡜ࡡ⛤ᗐࡡ࣐ࢪ࣏ࢴࢲࢅㆉืྊ⬗࠾࡞ࡗ࠷࡙ࠉ᭞࡞レ⣵࡝᳠ゞ࠿ᚪさ࡚࠵ࡾࠊ3.2 ࣁࢴࢠࢡࣚࢗࣤࢺࡡཋᅄ࡞ࡗ࠷࡙
ࠈTwo-pass TSA-FISH Ἢ࡛ 4 ⛸㢦ࡡࣈ࣭ࣞࣇࢅྜྷ᫤࡞⏕࠷ࡾࡆ࡛࡚ᵾⓏ㐿ఎᏄࢅ᳠ฝࡌࡾ஥࠿࡚ࡀࡒ࠿ࠉⳞ
మ࠿࣏ࢗࣤࢹࡈࡿ࡙࠷࡝࠷⟘ᡜ࠾ࡼࡡ㟸≁␏Ⓩ࡝⺧ක࠿ふᐳࡈࡿࡒ (Fig.1-B)ࠊࡐࡆ࡚ࠉࡆࡡ㟸≁␏Ⓩ࡝⺧කࡡཋᅄࢅ≁ᏽࡌࡾࡒࡴࠉtwo-pass TSA-FISH Ἢࢅ᧧షᕝ⛤ࡇ࡛࡞୯᩷ࡊࠉTSA ཬᚺࡡ Cy3 ỷ╌ࢅ⾔࠷ྊっ໩ࡊࡒᚃࠉ㢟᚜㙶࡞࡙ふᐳࡊࡒࠊࡐࡡ⤎ᯕࠉanti-DIG-HRP ࢅ⏕࠷ࡒୌᅂ┘ࡡᢘཋᢘమཬᚺᚃ࡞ࠉ᚜ᑚ࡚ࡢ࠵ࡾ࠿Ⳟమ࠿࣏ࢗࣤࢹࡈࡿ࡙࠷࡝࠷⟘ᡜ࠾ࡼࡡ㟸≁␏Ⓩ࡝⺧ක࠿ふᐳࡈࡿࡒࠊࡆࡿࡢࠉanti-DIG-HRP ࠿ࢪࣚ࢕ࢺ࡞㟸≁␏Ⓩ࡞ྺ╌ࡊࠉࡐࡿࢅ㉫Ⅴ࡛ࡊࡒࢨࢡࢻࣜቌᖕ࡞ࡻࡾࡵࡡ࡛᥆ᐳࡈࡿࡾࠊࡱࡒࠉࡆࡡᢘమࡡ㟸≁␏Ⓩ࡝ྺ╌࠿஦ᅂࡡ TSA ཬᚺ࡞ࡻࡽࠉ᭩⤂Ⓩ࡞㟸≁␏Ⓩ࡝ࣁࢴࢠࢡࣚࢗࣤࢺ࡞࡝ࡾ࡛⩻࠻ࡼࡿࡾࠊࡐࡆ࡚ࠉᢘཋᢘమཬᚺ᫤࡞࠽ࡄࡾཬᚺ᮪௲ࡡ᳠ゞ (ཬᚺࣁࢴࣆ࢒࣭ࡡ⤄ᠺࠉࣇࣞࢴ࢞ࣤࢡ᪁Ἢࠉཬᚺ᫤㛣ࠉᢘమ㔖࡝࡜) ࢅ⾔ࡖࡒ࠿ࠉ㟸≁␏Ⓩྺ╌ࢅよᾐࡌࡾࡆ࡛ࡢ௑ࡡ࡛ࡆࢀ࡚ࡀ࡙࠽ࡼࡍࠉᮇἪ࡞࠽ࡄࡾㄚ㢗ࡡୌࡗ࡛࡝ࡖ࡙࠷ࡾࠊ
4. ࡱ࡛ࡴࠈᮇ◂✪࡚ࡢࠉtwo-pass TSA-FISH Ἢࢅ⏕࠷ࡾࡆ࡛࡚᚜ᰶ⏍∸ࡡࢨࣤࢡࣜࢤࣅ࣭㐿ఎᏄ࠿᳠ฝྊ⬗࠾᳠ゞࡊࡒࠊࡐࡡ⤎ᯕࠉTSA-FISH Ἢ࡚ࡢវᗐ୘㊂࡚᳠ฝ࡚ࡀ࡝࠾ࡖࡒ࠿ࠉtwo-pass TSA-FISH Ἢࢅ⏕࠷ࡾࡆ࡛࡚ᵾⓏ㐿ఎᏄࢅ᳠ฝࡌࡾࡆ࡛࠿࡚ࡀࡒࠊࡊ࠾ࡊ࡝࠿ࡼࠉධ࡙ࡡⳞమ࠾ࡼ⺧කࢅᚋࡾࡆ࡛࠿࡚ࡀࡍࠉࡱࡒ㟸≁␏Ⓩ࡝
ࣁࢴࢠࢡࣚࢗࣤࢺࡵふᐳࡈࡿࡒࠊ௑ᚃࠉࡆࡿࡼࡡၡ㢗ࢅよỬࡌࡾࡆ࡛࡚ two-pass TSA-FISH Ἢࡢࠉin situ ࠾ࡗࢨࣤࢡࣜࢬ࡚ࣜࣝ࣊ࣜ㐿ఎᏄࢅྊっ໩࡚ࡀࡾᙁງ࡝ࢵ࣭ࣜ࡞࡝ࡾ࡛⩻࠻ࡼࡿࡾࠊ