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°¡. Áö³ð ¿£Áö´Ï¾î¸µ ±â¼úµé
1. Transcriptional activator-like effector genome editing
±×¸² 1. Transcriptional activator-like effector nuclease¸¦ ÀÌ¿ëÇÑ genome engineering (http://en.wikipedia.org/wiki/Tal_effector_nuclease)
Áö³ð¿£Áö´Ï¾î¸µÀÌ À¯ÀüÀÚ ¼¿ÀÇ Æ¯Á¤ºÎºÐÀ» ÀνÄÇÏ°í Ç¥ÀûÇÏ´Â °ÍÀ» ÀüÁ¦·Î Çϴ Ư¼º´öºÐ¿¡ zinc-finger(ZF)³ª transcriptionl activator-like(TAL) effector¿Í °°Àº DNA-°áÇÕ ´Ü¹éÁúÀÌ ÇϳªÀÇ Áö³ð¿£Áö´Ï¾î¸µ ±â¼úÀÌ µÉ ¼ö ÀÖ¾ú´Ù. Á¦ÇÑÈ¿¼Ò¿Í °°Àº DNA º¯ÇüÈ¿¼Ò¿Í ¿¬°áµÈ DNA-°áÇÕ ´Ü¹éÁúÀº ƯÁ¤ DNA¼¿¿¡ °áÇÕÇÑ ÈÄ À߶óÁÜÀ¸·Î½á ÀÌÁß°¡´ÚÀý´Ü(double strand break; DSB)¸¦ Çü¼º ÇÒ ¼ö ÀÖ°í »óµ¿Àû ÀçÁ¶ÇÕ(homologous recombination)À̳ª ºñ»óµ¿ ¸»´Ü ºÀÇÕ(non-homologous end joining; NHEJ)À» ÅëÇÏ¿© mutationÀ» µµÀÔÇÒ ¼ö ÀÖ¾ú´Ù. Ãʱ⿡ °¢±¤À» ¹Þ¾Ò´ø ZF-nuclease´Â motif´ç ¾à 3°³ÀÇ base pairs (bp)¸¦ ÀνÄÇÒ ¼ö ÀÖ¾úÁö¸¸ motif¸¦ stackingÇÏ´õ¶óµµ »ó´ëÀûÀ¸·Î ³·Àº ƯÀ̼º°ú ½ÇÇè½Ç¼öÁØ¿¡¼ ¾ò±â¿£ ºñ½Ñ ´ÜÁ¡À¸·Î ÀÎÇØ ÃÖ±Ù¿¡´Â TALEN(TAL effector nuclease)¸¦ ÀÌ¿ëÇÏ¿© Áö³ð¿£Áö´Ï¾î¸µÀ» ÇÏ´Â Ãß¼¼ÀÌ´Ù. ºñ·Ï ÇÑ motif´ç ÇϳªÀÇ bp¸¦ ÀνÄÇÏ°í Å« size·Î ÀÎÇØ assembly°¡ º¹ÀâÇÑ ÆíÀÌÁö¸¸ ±ä DNA¼¿À» ÀνÄÇϱâ À§ÇÑ stackingÀÌ ºñ±³Àû ½±±â ¶§¹®¿¡ Áö³ð¿£Áö´Ï¾î¸µ ±â¼ú·Î ¸¹ÀÌ »ç¿ëµÇ°í ÀÖ´Ù. Iowa state universityÀÇ Yang`s ÆÀÀº ÃÖ±Ù TALEN¸¦ ÀÌ¿ëÇÏ¿© È¿¸ð(Saccharomyces cerevisiae)ÀÇ À¯Àüü¿¡ ÀÖ´Â 3°³ÀÇ À¯ÀüÀÚÁ¦°Å(gene knockout)¿¡ ¼º°øÇÏ¿´´Ù.6 23bp±îÁö Àνİ¡´ÉÇÑ TALENÀ» ÀÎÀ§ÀûÀ¸·Î Á¦ÀÛÇÏ¿© È¿¸ðÀÇ Æ¯Á¤ À¯ÀüÀÚ¸¦ Á¦°ÅÇÏ¿´°í ¿¹»óµÇ¾îÁö´Â Ç¥ÇöÇüÀ» ¾òÀ» ¼ö ÀÖ¾ú´Ù. ¶ÇÇÑ »óµ¿Àû ÀçÁ¶ÇÕÀ» ÅëÇÑ À¯ÀüÀÚ ±³Ã¼µµ 34%ÀÇ È¿À²À» º¸¿´´Ù. ÀÌ·¯ÇÑ TALEN È°¿ë Áö³ð¿£Áö´Ï¾î¸µÀº Àúºñ¿ë°ú ³ôÀº È¿À²·Î ¿øÇϴ ǥÇöÇüÀ» ¾òÀ» ¼ö ÀÖ´Â »õ·Î¿î ±â¹ýÀ¸·Î ÁøÇÙ »ý¹°ÀÇ À¯ÀüÀÚ º¯Çü¿¡ »ó´çÇÑ ¿ªÇÒÀ» ÇÒ °ÍÀ¸·Î ±â´ëµÇ¾îÁø´Ù.7 8
2. Genome editing using an engineered type II CRISPR system
±×¸² 2. (a,b) Mechanism of CRISPR system. (Philippe H et al, Science, 2010) (c,d) Human genome editing with
engineered type II CRISPR system. (Mali P et al, Science, 2013.)
¿øÇÙ»ý¹°ÀÇ ÀûÀÀ¸é¿ª½Ã½ºÅÛ ¿ª½Ã ÁÁÀº Áö³ð¿£Áö´Ï¾î¸µ µµ±¸°¡ µÉ ¼ö ÀÖ´Ù. ÆÄÁö°¨¿°(phage infection)À̳ª ¼¼Æ÷°£ Á¢ÇÕ(conjugation)¿¡ ÀÇÇØ ¼¼Æ÷³»·Î À¯ÀÔµÈ ¿Ü·¡ À¯ÀüÀÚ¿Í °áÇÕ ÇÒ ¼ö ÀÖ´Â RNA¿Í Cas9 nuclease°¡ º¹ÇÕü¸¦ ÀÌ·ï DSB¸¦ ¾ß±âÇÏ´Â CRISPR(clustered regularly interspaced short palindromic repeats)ÀÇ ¿ø¸®¸¦ ±âº»À¸·Î ÇÏ´Â »õ·Î¿î ¹æ¹ýÀÌ ¹Ù·Î ±× ¿¹ÀÌ´Ù. ÃÖ±Ù ÇϹöµå ¸ÞµðÄà ½ºÄðÀÇ Church`s ÆÀÀº CRISPRÀÇ ½Ã½ºÅÛÀ» ÀÌ¿ëÇÏ¿© Æ÷À¯µ¿¹°¼¼Æ÷(mammalian cell)¿¡¼ non-sense GFP¸¦ activeÇÑ ÇüÅ·Π¹Ù²Ù¾ú´Ù.9 Cas9 nuclease¿Í °áÇÕÇÒ ¼ö ÀÖ´Â gRNA(guide RNA)¿Í Ÿ°Ù À¯ÀüÀÚ¸¦ ÀνÄÇÏ´Â RNA¸¦ °áÇÕ½ÃÄÑ ¼¼Æ÷³»·Î »ðÀÔ½ÃÅ°°í ±×¿Í µ¿½Ã¿¡ ÄÚµ· ÃÖÀûÈµÈ Cas9À» Á¦ÀÛÇÏ¿© ¼¼Æ÷³»·Î µµÀÔ½ÃÅ°°Ô µÇ¸é RNA°¡ Ÿ°Ù DNA¼¿°ú RNA-DNA base pairingÀ» Çü¼ºÇÏ°í Cas9ÀÇ nucleaseÈ°¼ºÀÌ DSB¸¦ Çü¼ºÇÏ°Ô µÈ´Ù. ±×·Î ÀÎÇØ »óµ¿Àû ÀçÁ¶ÇÕÀÌ ÃËÁøµÇ°Ô µÇ¹Ç·Î ¿øÇÏ´Â À¯ÀüÀÚÇüÀ» µµÀÔÇÒ ¼ö ÀÖ°Ô µÈ´Ù. ´õ¿í ½Å±âÇÑ °ÍÀº Cas9 nuclease°¡ ¹ßÇöµÉ ¶§¿Í ±×·¸Áö ¾ÊÀ» ¶§ÀÇ »óµ¿Àû ÀçÁ¶ÇÕ È®·üÀÌ °ÅÀÇ 8¹è³ª Â÷ÀÌ°¡ ³´Ù´Â °ÍÀÌ´Ù. ÀÌ´Â Áö¼ÓÀûÀÎ Cas9 nucleaseÀÇ ¹ßÇöÀÌ »óµ¿Àû ÀçÁ¶ÇÕÀ» ÃËÁøÇÑ´Ù´Â °ÍÀ» ¾Ï½ÃÇϱ⵵ ÇÑ´Ù. ÀÌ·¯ÇÑ CRISPR ½Ã½ºÅÛÀº ³·°Ô´Â 2%¿¡¼ ³ô°Ô´Â 25%±îÁö À¯ÀüÀÚ º¯Çü È¿À²À» º¸¿´´Ù. ÀÌ °á°ú´Â RNA-guided editingÀº ºü¸£°í multiplexibleÇÑ Áö³ð¿£Áö´Ï¾î¸µ µµ±¸·Î½á °¡´É¼ºÀ» º¸¿©Áá°í ÀÌ¿¡ µû¶ó Ãß°¡ÀûÀÎ ¿¬±¸µéÀÌ ÁøÇàµÇ°í ÀÖ´Â Ãß¼¼ÀÌ´Ù.10
3. Trackable multiplex recombineering(TRMR) method
±×¸² 3. (a) Trackable multiplex recombineering(TRMR) method (b) Multiple strategy to rapidly generate cell mixtures with
defined genetic modification. (Warner JR et al, Nat Biotechnol, 2010)
¹Ì»ý¹° Áö³ðÀ» ÀÌÇØÇϱâ À§Çؼ´Â ¸¹Àº ¼öÀÇ library°¡ ÇÊ¿äÇÏ´Ù´Â °ÍÀ» °¨¾ÈÇÑ´Ù¸é ÀûÀýÇÑ À¯ÀüÀÚº¯ÀÌ(genetic variation)À» Á¦ÀÛÇÑ ÈÄ¿¡ ±×·ÎºÎÅÍ ÆÄ»ýµÇ´Â ¿¬±¸¸¦ ÇÏ´Â °ÍÀÌ ½Ã°£ÀûÀ¸·Î³ª ºñ¿ëÀûÀ¸·Î³ª ÈξÀ °æÁ¦ÀûÀ̶ó´Â °ÍÀº ´©±¸µµ ºÎÁ¤ÇÏÁö ¸øÇÒ °ÍÀÌ´Ù. ÃÖ±ÙÀÇ Áö³ðÇÐ(genomics)ÀÇ ¹ß´Þ°ú multiplex DNA ÇÕ¼º ±â¼ú, ±×¸®°í »óµ¿Àû ÀçÁ¶ÇÕ ±â¼úÀÇ ¹ß´ÞÀº ÀÌ·¯ÇÑ ÀϵéÀÌ °¡´ÉÇÏ°Ô ÇÏ°í ÀÖ´Ù. Áö³ðÇÐÀÇ ¹ß´ÞÀº À¯ÀüÀÚ Á¦°Å¿Í °°Àº ¹æ¹ýÀ» ÅëÇÏ¿© À¯ÀüÀÚÀÇ Æ¯¼ºÀ» ÆľÇÇϴµ¥ µµ¿òÀ» ÁÖ¾ú°í ÀÌ´Â À¯ÀüÀÚÀÇ ¿ªÈ°À» È®ÀÎÇÒ ¼ö ÀÖ´Â °·ÂÇÑ µµ±¸°¡ µÇ¾úÁö¸¸ ´Ù¼ö À¯ÀüÀÚµéÀÇ µ¹¿¬º¯ÀÌ¿¡ ÀÇÇØ ¾ß±âµÇ´Â º¹ÇÕÀûÀÎ º¯È¿¡¼ Ç¥ÇöÇüÀ» ¿¹ÃøÇÏ°í ºÐ¼®ÇÏ´Â °Í¿¡´Â ¾ÆÁ÷ ¾Æ½¬¿î ¼º°ú¸¦ º¸ÀÌ°í ÀÖ¾ú´Ù. ÃÖ±Ù °³¹ßµÈ TRMRÀº ±âÁ¸ÀÇ ¹®Á¦Á¡À» º¸¿ÏÇÏ°í ½±°í ºü¸£°Ô ´Ù¼öÀÇ µ¹¿¬º¯À̸¦ Á¦ÀÛÇÒ ¼ö ÀÖ´Â ¹æ¹ýÀ¸·Î ¼Ò°³µÇ¾ú´Ù. ´Ü 1Àϸ¸¿¡ Àüü ´ëÀå±ÕÀÇ À¯ÀüÀÚÀÇ 95%¸¦ º¯ÇüÇÏ¿´°í À̷κÎÅÍ ¿øÇϴ ǥÇöÇüÀÇ ¹Ì»ý¹°À» ¼±º°ÇÒ ¼ö ÀÖ¾ú´Ù.11 ±âÁ¸¿¡ ÈçÈ÷ »ç¿ëµÇ´ø on-off¹æ½ÄÀÌ ¾Æ´Ñ up-down¹æ½ÄÀ¸·Î À¯ÀüÀÚÀÇ ¹ßÇöÀ» Á¶ÀýÇÔÀ¸·Î½á Ÿ°Ù À¯ÀüÀÚ¿¡ ´ëÇØ º¸´Ù Æø ³ÐÀº ÀÌÇظ¦ Á¦°øÇÏ¿´´Ù. ÀÎÀ§ÀûÀ¸·Î ÇÕ¼ºµÈ dsDNA´Â rolling circle amplification¿¡ ÀÇÇØ ÁõÆøµÇ°í ¼¼Æ÷³»¿¡¼ Áö³ðÀ¸·Î »ðÀÔµÊÀ¸·Î½á Ÿ°Ù À¯ÀüÀÚÀÇ ¹ßÇöÀ» Á¶ÀýÇÏ°Ô µÇ´Âµ¥ À̶§ ¸¸µé¾îÁö´Â mutant library´Â ¼±º°°úÁ¤À» °ÅÄ¡¸é¼ ¿øÇϴ ǥÇöÇüÀÇ ¼¼Æ÷¸¸À» ¼±º°ÇÒ ¼ö ÀÖ´Ù. ÀÌ·¯ÇÑ °úÁ¤À» ÅëÇØ ´Ü ÀÏÁÖÀϸ¸¿¡ ¿øÇÏ´Â ÇüÁú¿¡ ¿µÇâÀ» ÁÖ´Â À¯ÀüÀÚµéÀ» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù. ´ë´Ù¼öÀÇ À¯ÀüÀÚµéÀº »õ·ÎÀÌ ¹àÇôÁø À¯ÀüÀÚµéÀ̾úÀ¸¸ç ÀϺδ ±âÁ¸¿¡ ÀÌ¹Ì ¹àÇôÁ® ÀÖ´ø À¯ÀüÀÚ¿´°í ±× Áß¿¡¼´Â »õ·Î¿î ±â´ÉÀÌ ¹àÇôÁö±âµµ ÇÏ¿´´Ù. TRMRÀ» ÅëÇؼ ¹«¼öÈ÷ ¸¹Àº mutant library¸¦ Á¦ÀÛÇÒ ¼ö ÀÖÀ¸¸ç À̸¦ ÅëÇØ switches, oscillators ȤÀº sensors¿Í °°Àº Ãß°¡ÀûÀÎ ¿ªÇÒÀ» ÇÏ´Â À¯ÀüÀڵ鿡 ´ëÇؼµµ ±Ô¸íÇÒ ¼ö ÀÖÀ» °ÍÀÌ´Ù.12
4. Multiplex automated genome engineering
±×¸² 4. Rapid and continuous generation of sequence diversity by MAGE (Wang HH et al, Nature, 2009)
¸¹Àº Áö³ð¿£Áö´Ï¾î¸µ ±â¼úÀÌ ÇϳªÀÇ À¯ÀüÀÚ µ¹¿¬º¯ÀÌ¿¡ ÁÖ·Î ÃÊÁ¡À» ¸ÂÃá µ¥¿¡´Â ÀÌÀ¯°¡ ÀÖ´Ù. ZFNÀ̳ª TALEN, ±×¸®°í group II intron systemÀ» ÀÌ¿ëÇÑ ±â¹ýÀº ¸ðµÎ DSB¸¦ ¾ß±âÇϱ⠶§¹®¿¡ ´Ù¼öÀÇ À¯ÀüÀÚÀÇ µ¹¿¬º¯ÀÌ¿¡´Â ÀûÀýÄ¡ ¾Ê´Ù. ÀÌ·¯ÇÑ DSB°¡ ´Ù¼ö »ý¼ºµÇ°Ô µÈ´Ù¸é Àüü Áö³ðÀ» Æı«ÇÒ »Ó ¾Æ´Ï¶ó NHEJ¿¡ ÀÇÇØ ÀǵµÄ¡ ¾ÊÀº rearrangement¸¦ ¹ß»ý½ÃŲ´Ù. ¶ÇÇÑ ¥ë-red system¿¡ ÀÇÇØ ¸Å°³µÇ´Â dsDNA ÀçÁ¶ÇÕ ¿ª½Ã ³·Àº È¿À²À» º¸À̱⠶§¹®¿¡ ´Ù¼öÀÇ µ¹¿¬º¯ÀÌ Á¦ÀÛ¿¡´Â ÀûÀýÄ¡ ¾Ê´Ù. ÀÌ·¯ÇÑ ¹®Á¦Á¡À» º¸¿ÏÇϱâ À§Çؼ ÃÖ±Ù¿¡´Â dsDNA°¡ ¾Æ´Ñ ssDNA¸¦ ÀÌ¿ëÇÏ´Â ¹æ¹ýÀÌ ¼Ò°³µÇ¾ú´Ù. MAGE (multiplex automated genome engineering)Àº ÀÛÀº ssDNA°¡ lagging strandÀÇ Okazaki fragment ÇÕ¼º¿¡ ÇÁ¶ó¸Ó(primer)·Î ÀÛ¿ëÇÏ¿© µþ¼¼Æ÷ Áö³ð¿¡ µµÀÔµÇ¸é¼ À¯ÀüÀÚ º¯À̸¦ À¯µµÇÑ´Ù.13 MAGE´Â ssDNA¸¦ ¼¼Æ÷³»·Î µµÀÔ½ÃÅ´À¸·Î½á ¾ÆÁÖ °£´ÜÇÏ°Ô ´Ù¼öÀÇ µ¹¿¬º¯À̸¦ Á¦ÀÛÇÒ ¼ö ÀÖ¾ú´Ù. ¾à 30~100bpÀÇ ssDNA´Â »óµ¿ÀÇ ¸»´Ü¸¸ ÀÖÀ¸¸é Ÿ°Ù À¯ÀüÀÚ ºÎÀ§¿¡ »óº¸ÀûÀ¸·Î °áÇÕÇÔÀ¸·Î½á ¿ì¸®°¡ ¿øÇÏ´Â À¯ÀüÀÚ ¼¿À» µþ¼¼Æ÷·Î µµÀÔ½Ãų¼ö ÀÖ´Ù.14 ºñ½ÁÇÑ ¿ø¸®·Î MAGE´Â mismatch repair¿¡ °ü¿©ÇÏ´Â mutS À¯ÀüÀÚ¸¦ Á¦°Å½ÃÅ´°ú µ¿½Ã¿¡ ¥ë-red systemÀÇ ¹ßÇöÀ» ÅëÇØ ssDNA »ðÀÔÀ» 1000¹è±îÁöµµ Áõ°¡½Ãų¼ö ÀÖ¾ú´Ù. MAGE¿Í °°ÀÌ ssDNA-mediated recombinationÀÇ °æ¿ì ´Ù¸¥ plasmid³ª dsDNAº¸´Ù À¯ÀüÀÚ µµÀÔ(transformation)È®·üÀÌ ³ô±â ¶§¹®¿¡ º¸´Ù ³ôÀº È¿À²ÀÇ À¯ÀüÀÚ ÀçÁ¶ÇÕÀ» ±â´ëÇÒ ¼ö ÀÖÀ» »Ó¸¸ ¾Æ´Ï¶ó ÇÑ cycle´ç ¼Ò¿äµÇ´Â ½Ã°£ÀÌ »ó´ëÀûÀ¸·Î ´Ù¸¥ ±â¹ýµéº¸´Ù ª¾Æ ¸¹Àº ¼öÀÇ µ¹¿¬º¯ÀÌ ±ÕÁÖ¸¦ ¸¸µé ¼ö ÀÖ´Ù. ¶ÇÇÑ °£´ÜÇÑ ¿ø¸®´öºÐ¿¡ ´Ù¾çÇÑ ±ÕÁÖ¿¡¼µµ Àû¿ë°¡´ÉÇÒ °ÍÀ¸·Î ¿¹»óµÈ´Ù. ssDNA-mediated recombinationÀÇ ¸¹Àº ÀåÁ¡ ´öºÐ¿¡ ÇöÀçµµ ¸¹Àº ¿¬±¸µéÀÌ Ãß°¡ÀûÀ¸·Î ÁøÇàµÇ°í ÀÖ´Â »óȲÀÌ´Ù.15 16 17 18
³ª. ¹Ì¼¼À¯Ã¼ÀåÄ¡¸¦ ÀÌ¿ëÇÑ ¿ì¼ö ±ÕÁÖ ¼±º°
±×¸² 5. ´Ù¾çÇÑ ¹Ì¼¼À¯Ã¼ÀåÄ¡°¡ ÀÀ¿ëµÈ »ç·Êµé
´Ù¾çÇÑ ÇÕ¼º»ý¹°ÇÐÀû ±â¼úÀ» »ç¿ëÇؼ À籸¼ºµÈ »õ·Î¿î À¯Àüü´Â °¢°¢ÀÇ °³Ã¼µéÀÌ ¸ðµÎ ´Ù¸¥ È°¼ºÀ» °¡Áö°Ô ÇÑ´Ù. ¿¹¸¦ µé¾î À§¿¡ ¼³¸íµÈ MAGE¿Í °°Àº ¹æ¹ýÀ¸·Î À籸¼ºµÈ DNA¸¦ °¡Áø ±ÕÁÖµéÀÌ ¸¸µé¾îÁö¸é, À̸¦·ÎºÎÅÍ ¿øÇϴ ǥÇöÇüÀÇ ±ÕÁÖ¸¦ ¼±ÅÃÀûÀ¸·Î ºÐ¸®Çϱâ À§Çؼ´Â °íÀüÀû ¹æ¹ýÀ¸·Î´Â ¸¹Àº ³ëµ¿·Â°ú ½Ã°£ÀÌ ÇÊ¿äÇß¾ú´Ù. ÇÏÁö¸¸ ÃÖ±Ù ¹Ì¼¼À¯Ã¼°øÇÐ(microfluidics)µî°ú °°Àº »õ·Î¿î ±â¼úÀÌ µîÀåÇÏ¸é¼ Á» ´õ ¼Õ½±°í, ºü¸£°í È¿°úÀûÀ¸·Î °í¼Ó´ë·®½ºÅ©¸®´× (high-throughput screening; HTS)À» °¡´ÉÇÏ°Ô ÇÏ¿´´Ù. ÇÕ¼º»ý¹°Çаú´Â ¿ÏÀüÈ÷ ´Ù¸¥ ¿¬±¸ºÐ¾ßÀÎ ¹Ì¼¼À¯Ã¼°øÇÐÀÌ ÇÔ²² Çù¾÷(collaboration)µÇ¾î ¼º°øÀûÀϼö ÀÖ´Ù´Â °¡´É¼ºÀ» º¸¿©ÁÖ¾ú´ø »ç·Ê´Â 2005³â ½ºÅÄÆ÷µåÀÇ ÄùÀÌÅ© ¿¬±¸±×·ìÀÌ ÀÚµ¿ÈµÈ ¹Ì¼¼À¯Ã¼ÀåÄ¡ ³»¿¡¼ ¹Ì»ý¹°ÀÇ »ýÀåÀ» ÃËÁø, ÀúÇØ, À¯µµ µî°ú °°ÀÌ ´Ù¾çÇÑ ¹æ¹ýÀ» ÅëÇØ ¹«·Á 40ÀÏ°£ °üÂûÇß´ø °ÍÀÌ Àß ¾Ë·ÁÁ® ÀÖ´Ù.19 ¶ÇÇÑ, CITÀÇ Leadbetter ±×·ìÀº microfluidic ÀåÄ¡¸¦ ÀÌ¿ëÇØ ±âÁ¸¿¡ À¯ÀüÁ¤º¸°¡ ÀüÇô ¾Ë·ÁÁ®ÀÖÁö ¾Ê¾Ò´ø ¹Ì»ý¹° TraponemaÀÇ ´ë·® 106°³ÀÇ PCRÀ» Çس´Ù. ÀÌ´Â bioMEMs ±â¼úÀÇ ÀåÁ¡ÀÎ ´ë·®ÀÇ sample 󸮰¡´É´É·Â°ú ¹ÝÀÀ¿¡ ±Ø¼Ò·®ÀÇ chemical¸¸ÀÌ ÇÊ¿äÇÑ Á¡À» ½ÊºÐ»ì·Á, target DNA ÀÚ¼¼ÇÑ Á¤º¸¸¦ ¾ËÁö ¸øÇصµ ƯÁ¤ geneÀÇ Á¸Àç¿©ºÎ¸¦ °¡·Á³¾ ¼ö ÀÖ´Â PCRµîÀÇ ±â¼ú¿¡ Àû¿ë ÇÒ ¼ö ÀÖ´Ù´Â °ÍÀ» º¸¿©ÁØ´Ù.20
À§¿¡ ¼³¸íµÈ µÎ°¡Áö ¹Ì¼¼À¯Ã¼ ÀåÄ¡¸¦ ±âÁ¡À¸·Î ÇÑ ´Ù¾çÇÑ Áö³ð¿£Áö´Ï¾î¸µ±â¼úÀº ¹Ì¼¼À¯Ã¼ÀåÄ¡¸¦ ÀÌ¿ëÇÑ ±â¼úµé°ú ÇÔ²² ÃÖ±Ù±îÁö Àû¿ëµÇ¾îÁ® ¿Ô´Ù. ¿¹¸¦ µé¸é, °¡Àå ±âº»ÀûÀÎ PCR°ú °°Àº ±â¼úµéÀÌ ¹Ì¼¼À¯Ã¼ÀåÄ¡³»¿¡¼ ÀçÇöµÇ¾ú°í21. À§¿¡ ¼³¸íµÈ ÄùÀÌÅ© ±×·ìÀÇ bioreactor´Â ¿©ÀüÈ÷ ¸¹Àº °ü½ÉÀ» ¹Þ°í ÀÖÀ¸¸ç, DNA sequence22, ƯÁ¤µÈ ´Ü¹éÁúÀÇ °Ë·®, °ËÃâÀÌ °¡´ÉÇÑ ÀÚµ¿ÈµÈ ÀåÄ¡23, Å©±â³ª ´Ù¸¥ ¿ÜºÎ ¿äÀÎÀ» »ç¿ëÇÑ »ý¹°Ã¼ÀÇ separationÀ§ÇÑ ¹Ì¼¼À¯Ã¼ÀåÄ¡24, ´Ü½Ã°£³»¿¡ 107~9°³ÀÇ »ùÇà ºÐ¼®ÀÌ °¡´ÉÇÑ ±¸È¹ÈµÈ ¹Ì¼¼¹æ¿ï »ý¼º ¹× ÀúÀåÀåÄ¡25 µîµî ¿©·¯°¡Áö ¹æ¹ýµéÀÌ ÇÕ¼º»ý¹°ÇÐ ºÐ¾ß ¿¬±¸¿¡ È°¿ëµÇ°í ÀÖ´Ù.
±×¸² 6. High-throughput screening of strains (Jina Y et al, Nat commun, 2013)
ÀÌ·¯ÇÑ Áö³ð¿£Áö´Ï¾î¸µÀ» Á¢¸ñÇÑ ÇÕ¼º»ý¹°Çаú ´ë»ç°øÇÐÀº ±²ÀåÈ÷ ¸¹Àº platform ±ÕÁÖµéÀ» ¸¸µé¾î³Â°í ethanol26, higher alcohol27, fatty acids28 µîÀÌ ¸¹Àº ÀçÁ¶ÇÕ ±ÕÁÖµé·ÎºÎÅÍ »ý»êµÇ°í ÀÖ´Ù. ÃÖ±Ù¿¡´Â MAGE¿Í TRMR°ú °°ÀÌ ´ÙÁß¿£Áö´Ï¾î¸µ ±â¼úÀÌ ¹ß´ÞÇÔ¿¡ µû¶ó ¼ö¸¹Àº ¹Ì»ý¹°±ºÁý¿¡¼ ÀûÀýÇÑ ±ÕÁÖ¸¦ ¼±º°Çϱâ À§ÇÑ ½ºÅ©¸®´× ±â¼úÀÌ ÇÊ¿äÇÏ°Ô µÇ¾ú´Ù. ÇÏÁö¸¸ Çü±¤¹°ÁúÀ» ÅëÇÑ Æ¯Á¤ ±ÕÁÖ ¼±º°29À̳ª enrichment¿Í °°Àº ¼±ÅþÐ(selective pressure)À» »ç¿ëÇÏ´Â ±âÁ¸ÀÇ HTS30´Â ¸¹Àº ½Ã°£ÀÌ ¼Ò¸ðµÈ´Ù´Â ´ÜÁ¡À» °¡Áö°í ÀÖ´Ù. ÀÌ¿¡ µû¶ó »ý¹°ÇÐÀû ½ºÅ©¸®´×º¸´Ù Á» ´õ ½±°í ºü¸¥ ¹æ¹ýÀ¸·Î bioMEMs°¡ ¶°¿À¸£°í ÀÖ°í °ü·Ã ¿¬±¸°¡ ¼öÇàµÇ°í ÀÖ´Ù. ÀÌó·³ Áö³ð ¿£Áö´Ï¾î¸µÀº ÇÕ¼º»ý¹°ÇÐ ¹× ´ë»ç°øÇÐÀÇ ¹ßÀüÀ» À§ÇÑ Ãæ½ÇÇÑ µµ±¸·Î½á ±×¸®°í bioMEMs´Â »õ·Î¿î ÆÄÆ®³Ê·Î½á Àΰø¹Ì»ý¹°ÀÇ °³¹ß¿¡ Áß¿äÇÑ ¿ªÇÒÀ» ÇÏ°í ÀÖ´Ù.
Âü°í¹®Çå
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