ㄽ
ㄽ ᩥ ᑂ ᰝ ࡢ せ ᪨
༤ኈࡢᑓᨷศ㔝ࡢྡ⛠ ༤ ኈ㸦 ⌮Ꮫ 㸧 Ặྡ ᆏ ᰿ ♸ ே Ꮫᤵࡢせ௳ Ꮫつ๎➨4 ᮲➨ 1࣭2 㡯ヱᙜ ㄽᩥ㢟┠Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis using genome editing technology
㸦ࢿࢵࢱࢶ࣓࢚࢞ࣝ࠾ࡅࡿࢤࣀ࣒⦅㞟ᢏ⾡ࢆ⏝࠸ࡓ⏥≧⭢࣍ࣝࣔࣥཷᐜయșࡢᶵ⬟ゎᯒ㸧 ㄽᩥᑂᰝᢸᙜ⪅ ᰝ ᩍ ᤵ ᒣᮏ ༟ ᑂᰝጤဨ ᩍ ᤵ ฟ ༤ ᑂᰝጤဨ ᩍ ᤵ ᆏᮏ ᩔ ᑂᰝጤဨ ᩍᤵ ᆏᮏ ᑦ ⏥≧⭢࣍ࣝࣔࣥ㸦TH㸧ࡣ⬨᳝ື≀ࡢⓎ⏕ࡸᡂ㛗ࠊ௦ㅰ࡞࠾࠸࡚㔜せ࡞ᙺࢆࡶࡗ࡚ ࠾ࡾࠊࡑࡢస⏝ࡣ2 ࡘࡢ⏥≧⭢࣍ࣝࣔࣥཷᐜయ㸦TRȘ࠾ࡼࡧ TRș㸧ࢆࡋ࡚㉳ࡇࡿࡇ ࡀ▱ࡽࢀ࡚࠸ࡿࠋTR ࡣ᰾ෆཷᐜయࢫ࣮ࣃ࣮ࣇ࣑࣮ࣜᒓࡍࡿ㌿ᅉᏊ࡛࠶ࡾࠊTH ᛂ⟅ 㑇ఏᏊࡢ㌿ࢆTH ౫Ꮡⓗಁ㐍ࡲࡓࡣᢚไࡍࡿࠋ୧⏕㢮ࡣኚែ࠸࠺ࣘࢽ࣮ࢡ࡞Ⓨ⏕ᵝ ᘧࢆ᭷ࡋ࡚࠾ࡾࠊTH ౫Ꮡⓗᗂ⏕⤌⧊ࢆᾘኻ࠶ࡿ࠸ࡣᡂయ⤌⧊ᵓ⠏ࡍࡿࡇࡼ ࡗ࡚ࠊᗂ⏕ᆺࡽᡂయᆺయไࢆኚࡉࡏࡿࠋࡇࢀࡲ࡛୧⏕㢮ࡢࣔࢹࣝ⏕≀࡛࠶ࡿࢶ ࣓࢚࢞ࣝ࠾࠸࡚ࠊኚែ㛵㐃ࡍࡿTH ᛂ⟅㑇ఏᏊࡀᩘከࡃྠᐃࡉࢀ࡚ࡁࡓࡀࠊࡇࢀࡽࡢ 㑇ఏᏊᶵ⬟ゎᯒࡣࢇ㐍ࢇ࡛࠸࡞࠸ࠋࡑࡢ⌮⏤ࡋ࡚ࠊ୧⏕㢮࠾ࡅࡿᐇ⏝ⓗ࡞ᶆⓗ 㑇ఏᏊ◚ቯἲࡀ☜❧ࡉࢀ࡚࠸࡞ࡗࡓࡇࡀᣲࡆࡽࢀࡿࠋࡑࡇ࡛ࠊᮏ◊✲࡛ࡣࠊࢶ࣓࢚࢞ ࣝ࠾࠸࡚ࢤࣀ࣒⦅㞟ᢏ⾡ࢆ⏝࠸ࡓ㑇ఏᏊ◚ቯἲࢆ☜❧ࡋࠊኚែ㔜せ⪃࠼ࡽࢀࡿtrș 㑇ఏᏊࡢᶵ⬟ゎᯒࢆ⾜࠺ࡇࢆ┠ⓗࡋࡓࠋ ➨㸯❶࡛ࡣࠊࣇࣜ࢝ࢶ࣓࢚࢞ࣝ࠾ࡅࡿ㑇ఏᏊ◚ቯἲࡢ☜❧ࢆ⾜ࡗࡓࠋࣇࣜ࢝ࢶ࣓ ࢚࢞ࣝࡣྂࡃࡽ⏝࠸ࡽࢀ࡚࠸ࡿ୧⏕㢮ࡢࣔࢹࣝ⏕≀࡛࠶ࡿࡀࠊ␗㉁4 ಸయ࡛࠶ࡿࡓࡵ 㑇ఏᏊᶵ⬟ゎᯒࡣᶵ⬟㛗ᛶࡢ࠶ࡿ2 ࡘࡢ┦ྠ㑇ఏᏊ㸦࣓࣍࢜ࣟࢢ㸧ࢆྠ◚ቯࡍࡿ
ᚲせࡀ࠶ࡗࡓࠋࡑࡇ࡛ࠊேᕤ DNA ษ᩿㓝⣲࡛࠶ࡿ transcription activator-like effector nuclease㸦TALEN㸧ࢆ⏝࠸࡚㑇ఏᏊ◚ቯࢆヨࡳࡓࠋTALE ࣜࣆ࣮ࢺࡢ࣑ࣀ㓟ࣂ࢚࣮ࣜࢩ
ࣙࣥὀ┠ࡋࠊࡑࢀࡽࢆ࿘ᮇⓗ⤌ࡳྜࢃࡏ ࡓᚑ᮶ᆺࡼࡾ㧗࠸άᛶࢆ♧ࡍ TALEN
(Platinum TALEN)ࢆ⏝࠸࡚ࠊⰍ⣲ྜᡂ㛵㐃ࡍࡿtyr㑇ఏᏊࡢ࣓࣍࢜ࣟࢢ㸦tyr.L, tyr. S㸧
egfp 㑇ఏᏊࡢ」ᩘ㑇ఏᏊྠ◚ቯ࠾ࡼࡧ npm3 㑇ఏᏊࡢ࣓࣍࢜ࣟࢢྠ◚ቯᡂຌࡋ ࡓࠋࡇࢀࡽࡢ⤖ᯝࡽࠊࣇࣜ࢝ࢶ࣓࢚࢞ࣝ࠾࠸࡚TALEN ࢆ⏝࠸ࡓ㑇ఏᏊ◚ቯࡀ᭷ຠ ࡛࠶ࡿࡇࢆ♧ࡋࡓࠋ ➨㸰❶࡛ࡣࠊࢿࢵࢱࢶ࣓࢚࢞ࣝ࠾ࡅࡿ㑇ఏᏊ◚ቯἲࡢ☜❧ࢆヨࡳࡓࠋࢿࢵࢱࢶ࣓ ࢚࢞ࣝࡣ2 ಸయ࡛࠶ࡿࡇࡸࠊᛶᡂ⇍せࡍࡿ㛫ࡀ▷࠸ࡇ࡞ࡢⅬࡽࠊࣇࣜ࢝ ࢶ࣓࢚࢞ࣝࡼࡾࡶ㑇ఏᏊ◚ቯἲࢆ࣮࣋ࢫࡋࡓ㑇ఏᏊᶵ⬟ゎᯒ㐺ࡋࡓࣔࢹࣝ⏕≀࡛࠶ ࡿ ࠋclustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas)ࢩࢫࢸ࣒ࡣࠊᵓ⠏ࡢ⡆౽ᛶ㎿㏿ᛶຍ࠼࡚ࠊCas9 ࢾࢡ
࣮ࣞࢮࢆ mRNA ࡢ௦ࢃࡾࢱࣥࣃࢡ㉁ࡋ࡚ᑟධ࡛ࡁࡿࡇࡽࠊࡼࡾ᪩ᮇ㧗ຠ⋡ ࡞㑇ఏᏊ◚ቯࡀᮇᚅ࡛ࡁࡿ⪃࠼ࡓࠋࡑࡇ࡛ࡲࡎࠊⰍ⣲ྜᡂ㛵㐃㑇ఏᏊ㸦tyr, oca2, tyrp1, slc45a2㸧║ᙧᡂ㛵㐃㑇ఏᏊ㸦pax6㸧ࡢ◚ቯࢆࣔࢹࣝࠊsgRNA Cas9 ࢾࢡ࣮ࣞࢮ ࢆࣜ࣎ࢾࢡࣞ࢜ࢱࣥࣃࢡ㉁㸦RNP㸧ࡋ࡚ࢿࢵࢱࢶ࣓࢚࢞ࣝཷ⢭༸ᑟධࡋࠊ㧗ຠ⋡࡞ 㑇ఏᏊ◚ቯࡢ᮲௳᳨ウࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊࣇ࢘ࣥࢲ࣮㸦F0㸧ୡ௦࡛㠀ᖖ㧗࠸ຠ⋡࡛ 㑇 ఏ Ꮚ ◚ ቯ ࡢ ⾲ ⌧ ᆺ ಶ య ࢆ స ฟ ࡛ ࡁ ࡿ ᮲ ௳ ࢆ タ ᐃ ࡍ ࡿ ࡇ ᡂ ຌ ࡋ ࡓ ࠋ ࡇ ࢀ ࡽ CRISPR-Cas ࢩࢫࢸ࣒ࢆ⏝࠸࡚స〇ࡋࡓ㑇ఏᏊ◚ቯಶయ㸦௨ୗࠊcrispant ࡪ㸧ࡢᶆⓗ ࢤࣀ࣒㡿ᇦࢆḟୡ௦ࢩ࣮ࢡ࢚ࣥࢧ࣮ࡼࡾゎᯒࡋࡓࡇࢁࠊ㠀ᖖ㧗࠸ኚ␗ᑟධຠ⋡㸦య ⣽⬊ኚ␗⋡99㸣௨ୖ㸧࡛࠶ࡿࡇࡀศࡗࡓࠋࡇࢀࡽࡢᡂᯝࡽࠊCRISPR-Cas ࡢ RNP ᑟධࡣࠊࢿࢵࢱࢶ࣓࢚࢞ࣝࡢ F0 ୡ௦࠾࠸࡚⡆౽ࡘ㧗ຠ⋡࡞㑇ఏᏊ◚ቯἲ࡛࠶ࡿࡇ ࡀ♧ࡉࢀࡓࠋ ➨3 ❶࡛ࡣࠊࢿࢵࢱࢶ࣓࢚࢞ࣝ࠾ࡅࡿ⏥≧⭢࣍ࣝࣔࣥཷᐜయș(TR)ࡢᶵ⬟ゎᯒࢆ⾜ ࡗࡓࠋTR ࡣࠊ୧⏕㢮ࡢኚែ࠾ࡅࡿ TH ࢩࢢࢼࣝఏ㐩⤒㊰ࡢ᭱ୖὶ⨨ࡍࡿࠋ≉ࠊ TRșࡣ⏕యෆࡢ TH ⃰ᗘࡀୖ᪼ࡍࡿ࣏ࢪࢸࣈࣇ࣮ࢻࣂࢵࢡไᚚࡼࡗ࡚ࡑࡢⓎ⌧ ࡀⓗୖ᪼ࡍࡿࡇࡽࠊኚែࡢ࣐ࢫࢱ࣮ไᚚᅉᏊࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊ➨ 2 ❶࡛☜❧ࡋࡓ CRISPR-Cas ࢩࢫࢸ࣒ࢆ⏝࠸࡚ trș 㑇ఏᏊ◚ቯಶయ㸦trș crispant㸧ࢆ స〇ࡋࠊኚែ࠾ࡅࡿTRșࡢᶵ⬟ゎᯒࢆ⾜ࡗࡓࠋtrș㑇ఏᏊࡢࢥ࣮ࢻ㡿ᇦ࠾ࡼࡧࢫࣉࣛ ࢫࢻࢼ࣮ࢧࢺࢆᶆⓗࡋࡓsgRNA ࢆస〇ࡋࠊsgRNA/Cas9 RNP ࡋ࡚ཷ⢭༸ᑟධࡋ ࡓࡇࢁࠊᶆⓗ㓄ิࡢኚ␗ᑟධࡼࡿ㑇ఏᏊ◚ቯ࠾ࡼࡧࢫࣉࣛࢩࣥࢢ࢚࣮ࣛࡀ☜ㄆࡉ ࢀ ࡓ ࠋ ḟ TH ࡢ ᛂ ⟅ ⬟ ࢆ ㄪ ࡿ ࡓ ࡵ ࠊ ኚ ែ ๓ ᮇ ࡢ ࢜ ࢱ ࣐ ࢪ ࣕ ࢡ ࢩ TH 㸦Triiodothyronine; T3㸧ࢆᭀ㟢ࡋࡓࡇࢁࠊࢥࣥࢺ࣮ࣟࣝಶయ࡛ࡣᚋ⫥ࡢⓎ㐩ࡸ࢚ࣛࡢ㏥ ⦰࠸ࡗࡓ㢧ⴭ࡞ኚែ⌧㇟ࡀㄏᑟࡉࢀࡓࡀࠊtrș crispant ࡛ࡣ࢚ࣛࡢ㏥⦰ࡀᢚไࡉࢀࡓࠋ T3 ᭚㟢ಶయ࠾ࡅࡿ TH ᛂ⟅㑇ఏᏊࡢⓎ⌧ࢆࣜࣝࢱ࣒ RT-PCR ἲ࡛ゎᯒࡋࡓࡇࢁࠊ trș crispant ࡛ࡣ㌿ᅉᏊ࡛࠶ࡿ klf9 ࡸ fra-2 ࡢⓎ⌧ࡀಁ㐍ࡉࢀࡓࠋࡲࡓࠊ⮬↛ኚែ࠾ ࡅࡿtrș㑇ఏᏊḞᦆࡢᙳ㡪ࢆㄪࡓࡇࢁࠊtrș crispant ࡣࢥࣥࢺ࣮ࣟࣝಶయẚ㍑ࡋ࡚ ᑿࡢ㏥⦰ࡀ᭷ព㐜ᘏࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡋࡋ࡞ࡀࡽࠊtrș crispant ࡢᑿࡣ ྾ࡉࢀࠊ⮬↛ኚែࢆࡋࡓࠋࡲࡓࠊᗂ⏕≉ᚩⓗ࡞ゐゅࡢ㏥⦰ࡶྠᵝ㐜ᘏࡋࡓࠋ ୍᪉ࠊኚែ㝿ࡋ࡚ᵓ⠏ࡉࢀࡿᑠ⭠࡛ࡣ trș crispant ࠾࠸࡚ࡶᡂయ≉ᚩⓗ࡞ࣄࢲ ᵓ㐀ࡀほᐹࡉࢀࡓࠋࡉࡽࠊtrș crispant ྠኈࢆࡅྜࢃࡏࡓ F1 ୡ௦ࡢ trșኚ␗య࡛ࡶࠊ F0 ಶయྠᵝ࡞ኚែࡀほᐹࡉࢀࡓࠋ ௨ୖࡢ⤖ᯝࡽࠊࣇࣜ࢝ࢶ࣓࢚࢞ࣝࢿࢵࢱࢶ࣓࢚࢞ࣝࡢ୧✀࠾࠸࡚ࡇࢀࡲ࡛ᅔ 㞴࡛࠶ࡗࡓ㑇ఏᏊ◚ቯࢆ࣮࣋ࢫࡋࡓ㑇ఏᏊᶵ⬟ゎᯒἲࡢ☜❧ᡂຌࡋࡓࠋࡉࡽࡇࡢᢏ ⾡ࢆ⏝࠸࡚ࠊࢿࢵࢱࢶ࣓࢚࢞ࣝࡢኚែ࠾ࡅࡿTRșࡢᶵ⬟ࢆ♧၀ࡍࡿ⤖ᯝࡀᚓࡽࢀࡓࠋ ࡇࢀࡽࡢ◊✲ᡂᯝࡣࠊ୧⏕㢮ኚែࡢศᏊᶵᵓ࠾ࡼࡧTH ࡢⓎ⏕࣭ᡂ㛗ࡢᙺࡢゎ᫂㔜 せ࡞▱ぢࢆࡶࡓࡽࡍ◊✲ࡋ࡚㧗ࡃホ౯ࡉࢀࡿࠋ ௨ୖࠊᑂᰝࡢ⤖ᯝࠊᮏㄽᩥࡢⴭ⪅ࡣ༤ኈ㸦⌮Ꮫ㸧ࡢᏛࢆᤵࡉࢀࡿ༑ศ࡞㈨᱁ࡀ࠶ࡿ ࡶࡢㄆࡵࡿࠋ
බ⾲ㄽᩥ
Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases..
Yuto Sakane, Tetsushi Sakuma, Keiko Kashiwagi, Akihiko Kashiwagi, Takashi Yamamoto and Ken-ichi T. Suzuki.
Development, Growth & Differentiation 56: 108-114 (2014)
Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas.
Yuto Sakane, Midori Iida, Takashi Hasebe, Satoshi Fujii, Daniel R. Buchholz, Atsuko Ishizuya-Oka, Takashi Yamamoto and Ken-ichi T. Suzuki.
Biology Open 7 (2018) ཧ⪃ㄽᩥ
Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Tetsushi Sakuma, Hiroshi Ochiai, Takehito Kaneko, Tomoji Mashimo, Daisuke Tokumasu, Yuto Sakane Y, Ken-ichi Suzuki, Tatsuo Miyamoto, Naoaki Sakamoto, Shinya Matsuura and Takashi Yamamoto.
