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Home > ÀüÁ¦Ç°º¸±â > Inducible System ¡¤ Yeast hybrid > Tet À¯µµ ¹ßÇö ½Ã½ºÅÛ > Tet-One Inducible Expression (One vector System)
All-in-one vector ÇüÅÂÀÇ Tet-inducible system

Tet-One Inducible Expression (One vector System)

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Tet-One Inducible Expression System
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Lenti-X¢â Tet-One¢â Inducible Expression System
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Retro-X Tet-One Inducible Expression System
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Retro-X Tet-One Inducible Expression System (puro)
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AAVpro¢ç Tet-One Inducible Expression System (AAV2)
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  • °­·ÂÇÏ°Ô ¹ßÇöÀ» Á¶Àý ÇÒ ¼ö ÀÖ´Â Tet-On 3G systemÀ» ±â¹ÝÀ¸·Î ÇÑ all-in-one vectorÇüÅÂ
  • ³·Àº ¹é±×¶ó¿îµå¿Í ³ôÀº À¯µµ¹ßÇö È¿À²
  • 10 ng/mlÀÇ doxycycline¿¡µµ ¹ÝÀÀÇÏ´Â ¹Î°¨µµ
  • Lenti-X Tet-One: °í¿ª°¡ÀÇ ·»Æ¼¹ÙÀÌ·¯½º¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â ÆÐŰ¡ ½Ã½ºÅÛ Æ÷ÇÔ (Lenti-HTX Pakaging system)
  • Retro-X Tet-One: °í¿ª°¡ÀÇ ·¹Æ®·Î¹ÙÀÌ·¯½º¸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â ÆÐŰ¡ ½Ã½ºÅÛ Æ÷ÇÔ (Retro-X Universal Packaging System)
  • AAVpro Tet-One: helper-free packaging systemÀ» »ç¿ëÇϸç, AAV Particle ÃßÃâÀ» À§ÇÑ ÃßÃâ ½Ã¾à µîÀÌ Æ÷ÇԵǾîÀÖ´Ù.

Tet-One SystemÀÌ °³¹ßµÇ±â Àü¿¡´Â ClontechÀÇ Tet-on, Tet-off systemÀº response vector¿Í regulatory vector(doxycycline-responsive transactivator protein)ÀÎ 2°³ÀÇ vector¸¦ À¯ÀüÀÚ ¹ßÇöÀ» Á¶ÀýÇß´Ù.

Tet-One SystemÀº ÇϳªÀÇ º¤ÅÍ¿¡ ±¸¼º¿ä¼Ò¸¦ ¸ðµÎ Æ÷ÇÔÇÏ°í ÀÖ´Ù. Tet-On 3G transactivator´Â human phosphoglycerate kinase 1 promoter¸¦ ÀÌ¿ëÇÏ¿© Á¤¹æÇâÀ¸·Î ¹ßÇöµÇ¸ç, ¹Ý´ë ¹æÇâ¿¡´Â PTRE3GS promoter¸¦ ÀÌ¿ëÇÏ¿© ¸ñÀû À¯ÀüÀÚ(Gene of interest)ÀÇ ¹ßÇöÀ» Á¶ÀýÇÑ´Ù. ±âÁ¸¿¡ ¹ßÇ¥µÈ all-in-one vector systemÀº 50~100¹è Á¤µµÀÇ À¯µµ¹ßÇö È¿À²À» º¸ÀδÙ. ÇÏÁö¸¸ ClontechÀÇ Tet-One systemÀº all-in-one vector ÇüÅ·ΠÃÖ´ë 2500¹èÀÇ À¯µµ¹ßÇö È¿À²À» º¸ÀδÙ.(Heinz et al, 2011).


±×¸². Tet-One System Mechanism - The Tet-On 3G transactivator protein is expressed constitutively from the human PGK promoter (PPGK) but is unable to bind to the TRE3G promoter (PTRE3G) in the absence of doxycycline (Dox). When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds to PTRE3G and activates transcription of a transgene cloned downstream.


±×¸². Seven out of eight Tet-One clones show more than 1,000-fold induction of expression. HeLa cells were infected with LVX-TetOne-Puro-Luc lentivirus (M.O.I. = 1) and eight individual clones were selected and expanded according to the protocol. Each clone was analyzed for doxycycline-induced expression of luciferase. Seven of the eight clones demonstrated more than 1,000-fold induced expression, including two clones with 5,000-fold induction, and one clone with 10,000-fold induction.


±×¸². The Tet-One system is highly sensitive to low levels of doxycycline (Dox). HEK 293 cells were infected with LVX-TetOne-Luc lentivirus (M.O.I.=1) and the transduced pool was treated with 10-fold dilutions of doxycycline. 48 hr after treatment, the cells were harvested, lysed, and Western-blotted using anti-luciferase antibody. Luciferase expression reached maximum levels in the presence of only 10 ng/ml Dox.
References
Heinz, N., Schambach, A., Galla, M., Maetzig, T., Baum, C., Loew R., & Schiedlmeier, B. (2011) Retroviral and transposon-based Tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Human Gene Ther. 22(2):166-176.

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