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Tet-On 3G À¯µµ ¹ßÇö ½Ã½ºÅÛ

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
631164
Tet-On 3G Inducible Expression System (with ZsGreen1)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,062,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33594, x33597, x33151, x33200
Clontech
631165
Tet-On 3G Inducible Expression System (with mCherry)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,062,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33594, x99952, x33596, x99953, x33200, x33151
Clontech
631167
Tet-On 3G Inducible Expression System (EF1¥á Version)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,062,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33584, x33151, x33200
Clontech
631168
Tet-On 3G Inducible Expression System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,062,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33584, x33594, x33151, x33200
Clontech
631187
Lenti-X¢â Tet-On¢ç 3G Inducible Expression System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
each
(Package)
3,551,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32766, x32786, x33200
Clontech
631188
Retro-X Tet-On 3G Inducible Expression System
°ü·ÃÇмú ±¸¸ÅÇϱâ LMOÁ¦Ç°  ¶óÀ̼±½º 
Each
(Package)
3,168,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33346, x33548, x33557, x33558, x33014, x33200
Clontech
631348
Tet-On 3G Inducible Expression System (EF1alpha, ZsGreen1)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33594, x33597, x33151
Clontech
631337
Tet-On 3G Bidirectional Inducible Expression System
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,292,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33586, x33588, x33151
Clontech
631338
Tet-On 3G Bidirectional Inducible Expression System (with mCherry)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33588, x33589, x33151
Clontech
631339
Tet-On 3G Bidirectional Inducible Expression System (with ZsGreen1)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33302, x33588, x33590, x33151
Clontech
631340
Tet-On 3G Bidirectional Inducible Expression System (EF1alpha Version)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,292,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33586, x33588, x33151
Clontech
631341
Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, mCherry)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33588, x33589, x33151
Clontech
631342
Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, ZsGreen1)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33588, x33590, x33151
Clontech
631346
Tet-On 3G Inducible Expression System (EF1alpha, Bicistronic)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,292,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33592, x33594, x33151
Clontech
631347
Tet-On 3G Inducible Expression System (EF1alpha, mCherry)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
2,432,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x33362, x33594, x33596, x33151
Clontech
631363
Lenti-X¢â Tet-On¢ç 3G Inducible Expression System (EF1alpha Version)
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
Each
(Package)
3,551,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32766, x32786, x33200

  • DoxycyclineÀÇ À¯/¹«¿¡ µû¶ó À¯ÀüÀÚ ¹ßÇö (transcription)ÀÌ Á¶ÀýµÇ´Â inducible system
  • ¸Å¿ì ³·Àº Background¹ßÇö
  • Mammalian cell¿¡¼­ ƯÀ̼ºÀÌ ¸Å¿ì ³ôÀº À¯ÀüÀÚ ¹ßÇö À¯µµ °¡´É
Á¦Ç°¼³¸í
Tet-On¢çÀº doxycyclineÀÇ À¯/¹«¿¡ µû¶ó À¯ÀüÀÚ ¹ßÇö (transcription)ÀÌ Á¶ÀýµÇ´Â inducible systemÀÌ´Ù. ÀÌÁß Tet-On¢ç 3G tetracycline-inducible gene expression systemÀº 3¼¼´ëÀÇ Tet-On¢ç systemÀ¸·Î ´Ù¾çÇÑ ºÐ¾ß¿¡¼­ »ç¿ëµÇ°í ÀÖ´Ù. ÀÌ´Â 1¼¼´ë¿Í 2¼¼´ëÀÎ Tet-On¢ç °ú Tet-On¢ç Advanced system ´ëºñ background ¹ßÇöÀ» ³·Ãß°í tetracycline À¯»çüÀÎ doxycycline¿¡ ´ëÇÑ ¹Î°¨µµ¸¦ Çâ»ó½ÃŲ ±â¼úÀÌ´Ù.
Ç¥ 1
3¼¼´ë Tet-on À¯µµ ¹ßÇö ½Ã½ºÅÛÀº CMV promoter¸¦ »ç¿ëÇØ Tet-On¢ç 3G transactivator¸¦ ¹ßÇöÇÏ´Â ±âº» ½Ã½ºÅÛ Tet-On¢ç 3G Inducible Expression SystemÀ» Æ÷ÇÔÇÑ ´Ù¾çÇÑ ÇüÅ·ΠÆǸŵǰí ÀÖ´Ù.
  • Tet-On¢ç 3G Inducible Expression System (EF1alpha Version) (Code 631167 ¿Ü): EF1¥á promoter¸¦ »ç¿ëÇÏ¿© CMV promoter¿¡¼­´Â ½Ã°£ÀÌ Áö³¯¼ö·Ï ¹ßÇöÀÌ °¨¼ÒÇÏ´Â ¼¼Æ÷ (e.g. hematopoietic cells, stem cells)¿¡¼­µµ Tet-On¢ç 3G transactivator¸¦ ÀÏÁ¤ÇÏ°Ô Àå±â°£ ¹ßÇöÇÒ ¼ö ÀÖ´Ù.
  • Tet-On¢ç 3G Bidirectional Inducible Expression System (Code 631340 ¿Ü): Bidirectional promoter¸¦ »ç¿ëÇÏ¿© µÎ°¡Áö À¯ÀüÀÚ ¹ßÇöÀ» µ¿½Ã¿¡ À¯µµÇÒ ¼ö ÀÖ´Ù. À̶§ µÎ À¯ÀüÀÚ ¸ðµÎ µ¿µîÇÑ ¼öÁØÀ¸·Î ¹ßÇöµÇ¸ç À̸¦ ´Ù¾çÇÑ ÀÀ¿ë ºÐ¾ß¿¡¼­ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
    - °°Àº ¼¼Æ÷¿¡¼­ µÎ Á¾·ùÀÇ ¸ñÀû À¯ÀüÀÚ¸¦ µ¿½Ã ¹ßÇö
    - Á÷/°£Á¢ÀûÀÎ ÃøÁ¤ÀÌ ¾î·Á¿î ¸ñÀû À¯ÀüÀÚÀÇ ¹ßÇöÀ» ¸®Æ÷ÅÍ À¯ÀüÀÚÀÇ µ¿½Ã ¹ßÇöÀ¸·Î ¸ð´ÏÅ͸µ
    - ¸®Æ÷ÅÍ À¯ÀüÀÚÀÇ ¹ßÇöÀ» ¸ð´ÏÅ͸µÇÏ¿© À¯µµ ¹ßÇöÀÌ ³ô°í background°¡ ³·Àº clone ¼±º°
  • Tet-On¢ç 3G Inducible Expression System (with mCherry / with ZsGreen1 ¿Ü) (Code 631342 ¿Ü): IRES (internal ribosome entry site) ¼­¿­À» »ç¿ëÇØ ¸ñÀû À¯ÀüÀÚ¿Í Àû»ö (with mCherry) ȤÀº ³ì»ö (with ZsGreen1) Çü±¤ ´Ü¹éÁúÀ» °°ÀÌ ¹ßÇö½Ãų ¼ö ÀÖ´Ù. Çü±¤ ´Ü¹éÁú°ú ¸ñÀû À¯ÀüÀÚ´Â µ¿ÀÏÇÑ RNA Àü»çü¿¡¼­ º°µµ·Î µÎ ´Ü¹éÁú·Î À¯µµ ¹ßÇöµÇ¸ç ÀÌ Çü±¤ ´Ü¹éÁúÀ» »ç¿ëÇØ doxycycline ÷°¡ ½Ã ¸ñÀû À¯ÀüÀÚÀÇ À¯µµ ¹ßÇö È¿À²ÀÌ ³ôÀº cloneÀ» ¼±º°Çϴµ¥ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
  • Tet-One¢ç system (Code 634301 ¿Ü): ÀϹÝÀûÀ¸·Î »ç¿ëÇÏ´Â Tet-On¢ç 3G systemÀº Tet-On¢ç 3G transactivator¿Í tetracycline¿¡ ¹ÝÀÀÇÏ´Â À¯µµ ¹ßÇö promoter (PTRE3G)¸¦ °¢°¢ °¡Áö°í ÀÖ´Â 2 vector systemÀ̳ª, º» Á¦Ç°Àº 1 vector systemÀ¸·Î ½ÇÇèÀÇ ÆíÀǼºÀ» Áõ°¡½ÃÄ×´Ù.
  • PTRE3GV promoter¸¦ °¡Áø lentivirus¶Ç´Â retrovirus¸¦ »ý»êÇÏ´Â Lenti-X¢â/Retro-X¢â Tet-On¢ç 3G Inducible Expression SystemÀ» »ç¿ëÇϸé virus »ý»ê¿¡ ÇÊ¿äÇÑ viral packaging systemÀ» Çѹø¿¡ ÁغñÇÒ ¼ö ÀÖ´Ù.
³»¿ë
- °¢ Á¦Ç°ÀÇ »ç¿ëÀÚ ¸Å´º¾óÀ» Âü°íÇϼ¼¿ä.

ÁÖÀÇ) ÀÌ Á¦Ç°¿¡ Æ÷ÇÔµÈ pTRE vector´Â plasmid Á¦ÀÛ ½Ã ÀϹÝÀûÀÎ plasmidº¸´Ù ¼öÀ²ÀÌ ³·´Ù. µû¶ó¼­ ¼öÀ²À» ³ôÀ̱â À§ÇØ Æò¼Òº¸´Ù ¹è¾ç¾× ¾çÀ» ´Ã·Á¼­ »ç¿ëÇÏ´Â °ÍÀ» ÃßõÇÑ´Ù.
¶ÇÇÑ, ½ÃÁßÀÇ plasmid purification kit¸¦ »ç¿ëÇÏ´Â °æ¿ì low copy plasmid¿ë ÇÁ·ÎÅäÄÝÀ» »ç¿ëÇÏ´Â °ÍÀ» ±ÇÀåÇÑ´Ù.
Ư¡
- Bakcground °¨¼Ò: promoter ¼­¿­À» ÃÖÀûÈ­ÇÏ¿© ±âÁ¸ÀÇ PTIGHT ´ëºñ background ¹ßÇöÀ» 5~20¹è·Î °¨¼Ò½ÃÄ×´Ù. (±×¸² 1.)
- Dox¿¡ ´ëÇÑ ¹Î°¨µµ Áõ°¡: Tet-On¢ç 3G regulator vectorÀÇ ¼­¿­À» ÃÖÀûÈ­ÇÏ¿© doxycycline¿¡ ´ëÇÑ ¹Î°¨µµ°¡ ´ëÆø Áõ°¡µÇ¾î º¸´Ù ³·Àº ³óµµÀÇ Dox (5~10 ng/§¢)¿¡¼­µµ ¹ßÇöÀ» À¯µµÇÒ ¼ö ÀÖ´Ù. ´Ü 10 ng/§¢¸¦ ÇÏ·í¹ã ó¸®ÇÏ´Â °ÍÀ¸·Î ¸Å¿ì ³ôÀº ¹ßÇöÀ» À¯µµÇÒ ¼ö ÀÖ´Â °í°¨µµÀÇ ½Ã½ºÅÛÀÌ´Ù.
- °­ÇÑ ¹ßÇö À¯µµ ºñÀ²: Tet-On¢ç 3G´Â ±âÁ¸ÀÇ Tet-On ¹× Tet-Off system°ú °°ÀÌ ³ôÀº ÃÖ´ë ¹ßÇö ¼öÁØÀ» À¯ÁöÇϸ鼭µµ background¸¦ Å©°Ô ÁÙ¿´±â ¶§¹®¿¡ ¹ßÇö À¯µµ ½Ã ¸Å¿ì ³ôÀº ¹ßÇö À¯µµÀ²À» º¸Àδ٠(±×¸² 2). MCF7 ¼¼Æ÷¸¦ ÀÌ¿ëÇÑ ½ÇÇè¿¡¼­´Â ÃÖ´ë 27,000¹èÀÇ À¯µµ ºñÀ²ÀÌ È®ÀεǾú´Ù.

Tet System Approved FBS
Tet-On¢ç 3G systemÀº Tet System Approved FBS (Code 631107 ¿Ü)¿Í ÇÔ²² »ç¿ëÇÏ´Â °ÍÀÌ Áß¿äÇÏ´Ù. Tet-On¢ç 3G system doxycycline¿¡ ´ëÇÑ °¨µµ°¡ ¸Å¿ì Çâ»óµÇ¾ú±â ¶§¹®¿¡ tetracyclineÀÌ Æ÷ÇÔµÇÁö ¾ÊÀº FBS¸¦ »ç¿ëÇØ¾ß ÇÑ´Ù. Tet System Approved FBS (Code 631107 ¿Ü)´Â Tet-On¢ç 3G systemÀÇ background¹ßÇö¿¡ ¿µÇâÀ» ¹ÌÄ¡Áö ¾Ê´Â Á¡ÀÌ °ËÁõµÇ¾ú±â ¶§¹®¿¡ ÇØ´ç Á¦Ç°À» »ç¿ëÇÏ´Â °ÍÀ» ÃßõÇÑ´Ù. °¢ Tet-On¢ç 3G system¿¡´Â Tet System Approved FBS (Code 631107 ¿Ü) 50 §¢ÀÌ Æ÷ÇԵǾî ÀÖ´Ù.

±×¸² 1. PTRE3G ÇÁ·Î¸ðÅÍÀÇ ³·Àº background¹ßÇö
Tet-On¢ç 3G system°ú Tet-On¢ç Advanced systemÀ» »ç¿ëÇÏ¿© HEK293 ¼¼Æ÷¿¡ °¢°¢ regulatory vector¿Í response vector (luciferase)¸¦ transfection ÇÏ¿´´Ù. ÀÌÈÄ ¼¼Æ÷¸¦ Dox +/- (0, 50, 500 ng/§¢) »óÅ·Π24½Ã°£ ¹è¾ç ÈÄ luciferaseÀÇ ¹ßÇöÀ» ÃøÁ¤ÇÏ¿´´Ù. ±× °á°ú, Dox°¡ ¾ø´Â »óÅ¿¡¼­´Â Tet-On¢ç 3G system¿¡¼­´Â background ¹ßÇöÀÌ ¸Å¿ì ³·¾Ò°í (È®´ëµÈ ºÎºÐ), Dox°¡ ó¸®µÈ Á¶°Ç¿¡¼­´Â µÑ´Ù ¸Å¿ì °­ÇÑ ¹ßÇöÀ» º¸ÀÌ´Â °ÍÀ» È®ÀÎÇÒ ¼ö ÀÖ´Ù.

±×¸²2. ¹ßÇö À¯µµÀ² (induction rate)ÀÌ Çâ»óµÈ Tet-On¢ç 3G ½Ã½ºÅÛ
Tet-On¢ç, Tet-On¢ç Advanced, Tet-On¢ç 3G ¼¼°¡Áö ½Ã½ºÅÛÀ» »ç¿ëÇØ HEK 293 cell°ú HeLa cell¿¡ regulatory vector¿Í response vector (luciferase)¸¦ transfection ÈÄ 1 §¶/§¢ doxycyclineÀ¸·Î luciferase À¯ÀüÀÚ¸¦ ¹ßÇö½ÃÄ×´Ù. ÀÌÈÄ 18½Ã°£ ÀÌÈÄ 3¹Ýº¹À¸·Î luciferase È°¼ºÀ» ÃøÁ¤ÇÑ °á°ú Tet-On¢ç 3G systemÀÌ °¡Àå ³ôÀº ¹ßÇö À¯µµÀ²À» º¸ÀÌ´Â °ÍÀ» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.

±×¸²3. ¹Î°¨µµ°¡ Çâ»óµÈ Tet-On¢ç 3G ½Ã½ºÅÛ
GenomeÀÇ °°Àº À§Ä¡¿¡¼­Tet-On¢ç 3G ¿Í Tet-On¢ç AdvancedÀÇ µÎ °¡Áö ÇÁ·Î¸ðÅÍ¿¡ ÀÇÇØ ¾ÈÁ¤ÀûÀ¸·Î luciferase À¯ÀüÀÚ¸¦ ¹ßÇöÇÏ´Â HLF33 cell-line¿¡¼­ Dox ³óµµ¸¦ ¹Ù²ã°¡¸ç luciferaseÀÇ ¹ßÇö·®À» ÃøÁ¤ÇÏ¿´´Ù. ±× °á°ú 5~10 ng/§¢¿¡¼­´Â Tet-On¢ç 3GÀÇ cell-line¿¡¼­ 100~150¹è ³ô¾Ò°í, 50 ng/§¢¿¡¼­´Â ¹ßÇö·®ÀÌ 4.6¹è ³ô¾ÆÁ³´Ù.
(University of Erlangen, W. Hillen et. al)

±×¸²4. 10 ng/§¢ÀÇ Dox¿¡¼­µµ °í°¨µµ·Î ÀÛµ¿ÇÏ´Â Tet-On¢ç 3G

±×¸²5. ¸Å¿ì ³ôÀº S/N ratioÀÇ Tet-On¢ç 3G¸¦ »ç¿ëÇÑ ¼¼Æ÷ÁÖ
¼¼ Á¾·ùÀÇ Tet-On¢ç 3G cell-line¿¡ response vector (luciferase)¸¦ transfection Çß´Ù. ÀÌÈÄ Dox +/- »óÅ¿¡¼­ 24½Ã°£ ¹è¾ç ÈÄ luciferase È°¼ºÀ» ÃøÁ¤ÇÑ °á°ú, ¸ðµÎ ¼öõ¹èÀÇ S/N ratio¸¦ º¸¿´´Ù.
¿ëµµ
- Mammalian cell¿¡¼­ À¯ÀüÀÚ ¹ßÇö À¯µµ
- µ¶¼º À¯ÀüÀÚ ¹ßÇö Á¦¾î
- in vivo¿¡¼­ÀÇ ¸ñÀû À¯ÀüÀÚ ¹ßÇö À¯µµ
°ü·ÃÁ¦Ç°
- Any vector, any insert, any site·Î DNA assembly, In-Fusion¢ç Snap Assembly Master Mix (Code 638947)
- In-Fusion¢ç¿¡¼­ ÃÖ°íÀÇ cloning È¿À²À» À§ÇÑ Àü¿ë E.coli comp.cell, Stellar¢â Competent Cells (Code 636763)
- ´Ù¾çÇÑ cell¿¡ DNA / RNA / ´Ü¹éÁú ¸ðµÎ Àû¿ë °¡´ÉÇÑ transfection ½Ã¾à, TransIT-X2 Dynamic Delivery System (Code MIR 6000)
- ³» cell¿¡ ¸Â´Â MirusÀÇ ÃÖÀû transfection ½Ã¾à ã±â online tool, Reagent Agent¢ç
Cell Line Ãë±Þ ÁÖÀÇ»çÇ× (Ŭ¸¯)

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