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Gene transfer (virus)
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Retrovirus
> Retro-X Q vector
Retro-X Q vector
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5£§ LTRÀÇ promoter·Î ´Ù¸¥ retroivirus vectorº¸´Ù ³ôÀº ¿ª°¡¸¦ »ý»ê
Promoter interference Á¦°Å·Î ½Å·Ú¼º ³ôÀº ¹ßÇö°ú bicistronic ¹ßÇö
Çü±¤ ¸¶Ä¿¸¦ Æ÷ÇÔÇÏ¿© ´Ù¼öÀÇ ¼±º° ¸¶Ä¿
Retro-X Q Vector´Â °í¿ª°¡ÀÇ ¹ÙÀÌ·¯½º »ý»ê°ú ½Å·Ú¼º ³ôÀº ¹ßÇöÀ» ½ÇÇöÇϰí, promoter interferenceÀÇ °¡´É¼ºÀ» ÃÖ¼ÒÈ Çϵµ·Ï ¼³°èµÇ¾ú´Ù (1).
º» Q Vector´Â Ç×»ýÁ¦ ¶Ç´Â Çü±¤ ¼±º° ¸¶Ä¿ (Figure 1)¿Í ÇÔ²² ¸ñÀû À¯ÀüÀÚ°¡ ¹ßÇöµÇ°í, ¼÷ÁÖÀÇ genomeÀ¸·Î »ðÀÔµÈ ÈÄ 5£§LTRÀÇ promoter°¡ ºñȰ¼ºµÇµµ·Ï ¼³°èµÇ¾ú´Ù (Figure 2). Self-inactivating vector´Â promoter interference (2-5)ÀÇ °¡´É¼ºÀ» °¨¼Ò½ÃÄÑ ¹ßÇöÀ» Çâ»ó½ÃŲ´Ù. MMLV LTR¿¡¼ transcript¸¦ È¿°úÀûÀ¸·Î ¹ßÇöÇÏÁö ¸øÇÏ´Â ¼¼Æ÷¿¡¼µµ È®½ÇÇÑ ¹ßÇöÀ» º¸¿©ÁØ´Ù(6).
Figure 1. Composite Retro-X Q Vector Set map.
In this generalized version of the Q Vector Map, the common elements of all the vectors are represented. Immediately downstream of the CMV immediate early promoter, the multiple cloning site (MCS) is followed by a eukaryotic IRES that ensures a second ORF (an antibiotic resistance marker or another gene in the case of pQCXIX) is cotranscribed with the gene cloned into the MCS. The expression cassette has all of the essential elements for retroviral integration and expression.
Self-Inactivation Vector·Î ³ôÀº ¹ÙÀÌ·¯½º ¿ª°¡ »ý»ê.
5£§LTRÀÇ CMV/MSV hybrid promoter´Â packaging °úÁ¤ µ¿¾È ³ôÀº ¿ª°¡¸¦ À¯µµÇÑ´Ù. ±×·± ´ÙÀ½ ¼÷ÁÖ genomeÀ¸·Î »ðÀԵǴ µ¿¾È 3£§LTRÀÇ U3 ¿µ¿ªÀÇ °á½ÇÀÌ 5£§LTR¿¡ º¹Á¦µÇ¾î ºñȰ¼ºÈ½ÃŲ´Ù. µû¶ó¼ MCSÀÇ ¹Ù·Î »ó·ù¿¡ À§Ä¡ÇÑ ³»ºÎ CMV promoter¿¡ ÀÇÇØ¼¸¸ transcript°¡ ¹ßÇöµÈ´Ù. Vector set´Â 3°³ÀÇ Ç×»ýÁ¦ ¼±º° ¸¶Ä¿ (Hyg, Pur, Neo)¸¦ °¡Áö°í ÀÖÀ¸¸ç, À̵éÀº ¸ñÀû À¯ÀüÀÚ¿Í ÇÔ²² internal ribosome entry site (IRES)¸¦ ÅëÇÏ¿© bicistron Çü½ÄÀ¸·Î ¹ßÇöµÈ´Ù. LacZ control vector (pQCLIN) ¶ÇÇÑ ¼¼Æ®¿¡ Æ÷ÇԵǾî ÀÖ´Ù.
Figure 2. Self-inactivation mechanism of the Retro-X Q Vectors.
Plus strand viral RNA (blue) from the expression cassette is reverse transcribed. During integration, a circular intermediate is formed that results in duplication of the deletion in the U3 region of the 3' LTR. This inactivates the CMV/MSV hybrid promoter in the 5' LTR so that transcription can only be driven from the internal promoter, P
CMV IE
(Figure 1).
Components & Storage Conditions
°¢ Á¦Ç° ±¸¼º¹°°ú º¸°üÁ¶°ÇÀº Certificate of Analysis ¸¦ ÂüÁ¶ÇϽʽÿÀ.
References
1. Julius, M. A. et al. (2000) BioTechniques 28:702-708.
2. Emerman, M. & Temin, H. M. (1984) Cell 39:449-467.
3. Yee, J. K. et al. (1987) Proc. Natl. Acad. Sci. USA 84:5197-5201.
4. Nakajima, K. et al. (1993) FEBS Lett. 315:129-133.
5. Zufferey, R. et al. (1998) J. Virol. 72:9873-9880.
6. Soriano, P. et al. (1991) J. Virol. 65:2314-2319.
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