COVID-19 ¿¬±¸¸¦ À§ÇÑ Real Time PCR ½Ã¾à ¾È³»
ÃÖ±Ù ±ÞÁõÇÏ´Â Coronavirus SARS-CoV-2 (COVID-19) °¨¿°»ç·Ê·Î ºü¸£°í Á¤È®ÇÑ virus Áø´Ü¹ýÀÇ Çʿ伺ÀÌ ´ëµÎµÇ°í ÀÖ´Ù.
Takara Bio´Â viral RNA purification, NGS Á¦Ç°°ú RT-qPCR Á¦Ç°°ú
°°ÀÌ coronavirusÀÇ ÇмúÀû, ÀÓ»óÇÐÀû ¿¬±¸¸¦ À§ÇÑ ¼Ö·ç¼ÇÀ» Á¦°øÇÏ°í ÀÖ´Ù.
ƯÈ÷ COVID-19ÀÇ °í°¨µµ °ËÃâÀ» À§ÇØ, Takara BioÀÇ Real Time RT-PCR Á¦Ç°°ú È®¸³µÈ primer set¿Í ÇÔ²² »ç¿ëÇÏ¿© 5 copyÀÇ virus±îÁö °ËÃâÇÒ ¼ö ÀÖ´Ù (±×¸² 1).
´ÙÄ«¶ó ½Ã¾àÀÇ FDA ±ä±Þ»ç¿ë½ÂÀÎ (Emergency Use Authorizations) ÇöȲ
ISO 13485:2016 Á¶°Ç¿¡ µû¶ó Á¦Á¶µÈ °íÇ°ÁúÀÇ ´ÙÄ«¶ó ½Ã¾àÀº COVID-19 test¸¦ À§ÇØ ¹Ì±¹ FDA¿¡¼ ¹ßÇàÇÏ´Â ±ä±Þ»ç¿ë½ÂÀο¡ »ç¿ëµÇ°í ÀÖ´Ù.
±×¸² 1. TakaraÀÇ one-step Real Time RT-PCR Á¦Ç°À» ÀÌ¿ëÇÑ 5 ~ 50,000 copiesÀÇ COVID-19 °ËÃâ ¿¹
º» ½ÇÇè°á°ú´Â Áúº´ÅëÁ¦¿¹¹æ¼¾ÅÍ (CDC)¿¡¼ È®¸³µÈ ÇÁ·ÎÅäÄÝÀ» ÀÌ¿ëÇÏ¿© Takara Bio¿¡¼ ½ÇÇèÇÏ¿´À¸¸ç, º» µ¥ÀÌÅÍ´Â CDC¿¡ ½ÂÀιÞÁö´Â ¾Ê¾Ò´Ù.
»ýü½Ã·á·ÎºÎÅÍ Á÷Á¢ virus °ËÃâ - RNA Á¤Á¦°úÁ¤ ºÒÇÊ¿ä
ºÒÈ°¼ºÈ ó¸®µÈ Influenza A virus H1N1ÀÇ °ËÃâ ¿¹
PrimeDirect¢â Probe RT-qPCR Mix´Â º°µµÀÇ RNA Á¤Á¦°úÁ¤ ¾øÀÌ ´Ù¾çÇÑ »ýü½Ã·á¸¦ Á÷Á¢ ½ÇÇè¿¡ ÀÌ¿ëÇÒ ¼ö ÀÖ¾î ½Ã°£ ¹× ½Ã·á¸¦ ¾Æ³¥ ¼ö ÀÖ´Â ÀåÁ¡ÀÌ ÀÖ´Ù (±×¸² 2). Takara Bio´Â PrimeDirect Probe RT-qPCR Mix¸¦
ÀÌ¿ëÇÏ¿© ¾Æ·¡ÀÇ ½ÇÇè°úÁ¤¿¡ µû¶ó »ýü½Ã·á¿¡¼ Á÷Á¢ H1N1À» °ËÃâÇÏ¿´´Ù. ºÒÈ°¼ºÈ ó¸®ÇÑ Influenza A virus¸¦ ±¸°»óÇǼ¼Æ÷, ºñ°»ùÇÃ, Ÿ¾×¿¡ °¢°¢ 2 x 10
0 ~ 10
2 copies/§¡¸¦ ÷°¡ÇÏ¿´´Ù.
Å×½ºÆ® »ùÇà |
»ç¿ë¹æ¹ý |
±¸°»óÇǼ¼Æ÷ |
200 §¡ÀÇ PBS buffer¿¡ ±¸°»óÇǼ¼Æ÷ »ùÇÃÀ» äÃëÇÑ ¸éºÀÀ» ´ã±Å 5ºÐ°£ Èçµç ´ÙÀ½ »óÃþ¾× 1 §¡¸¦ PCR ¹ÝÀÀ¿¡ »ç¿ë |
ºñ°»ùÇà |
200 §¡ÀÇ PBS buffer¿¡ ºñ°»ùÇÃÀ» äÃëÇÑ ¸éºÀÀ» ´ã±Å 5ºÐ°£ Èçµç ´ÙÀ½ »óÃþ¾× 1 §¡¸¦ PCR ¹ÝÀÀ¿¡ »ç¿ë |
Ÿ¾× |
1 §¡¸¦ Á÷Á¢ PCR ¹ÝÀÀ¿¡ »ç¿ë |
Reagent |
Volume |
PrimeDirect Probe RT-qPCR Mix (2X) |
12.5 §¡ |
Forward Primer (10 ¥ìM) |
0.5 §¡ |
Reverse Primer (10 ¥ìM) |
0.5 §¡ |
qPCR probe (10 ¥ìM) |
0.5 §¡ |
Test sample |
1 §¡ |
Influenza A virus H1N1 (2 x 100 ~ 102 copies/§¡) |
1 §¡ |
RNase Free H2O |
9 §¡ |
Total |
25 §¡ |
±×¸² 2. PrimeDirect Probe RT-qPCR Mix¸¦ ÀÌ¿ëÇÏ¿© »ýü½Ã·á·ÎºÎÅÍ Á÷Á¢ Viral RNA¸¦ °ËÃâ.
ºñ°»ùÇà ¹× Ÿ¾×¿¡¼´Â 20 copies, ±¸°»óÇǼ¼Æ÷ »ùÇÿ¡¼´Â 2 copies ±îÁö ³ôÀº °¨µµ·Î °ËÃâµÇ¾ú´Ù.
COVID-19 °ËÃâ ÇÁ·ÎÅäÄÝÀÇ Primer/probe set¿ÍÀÇ È£È¯¼º È®ÀÎ
Takara Bio´Â ´ç»çÀÇ PrimeScript¢â III one-step RT-PCR mix¿Í PrimeDirect¢â Probe RT-qPCR Mix¸¦ ÀÌ¿ëÇÏ¿© COVID-19 °ËÃâ½ÇÇèÀ» ÁøÇàÇÏ¿´´Ù. À̶§ »ç¿ëµÈ Primer set´Â
¹Ì±¹°ú Áß±¹ÀÇ Áúº´ÅëÁ¦¿¹¹æ¼¾ÅÍ (CDC), WHO, ÀϺ» ±¹¸³°¨¿°Áõ¿¬±¸¼Ò (NIID) ·ÎºÎÅÍ È®¸³µÈ °ËÃâ¹ý¿¡ »ç¿ëÇÑ °Í°ú µ¿ÀÏÇÑ ¼¿ÀÌ´Ù. °ËÃâ°¨µµ´Â »ùÇÃÀÇ À¯Çü¿¡ µû¶ó ´Þ¶ú°í,
ÀϺ» NIID ÇÁ·ÎÅäÄÝÀº NIID·ÎºÎÅÍ Á¦°ø¹ÞÀº ÁÖÇü (template)À» »ç¿ëÇÏ¿´°í, ±× ¿Ü ½ÇÇ豺¿¡´Â Takara Bio¿¡¼ ÇÕ¼ºÇÑ RNA¸¦ »ç¿ëÇÏ¿´´Ù (ÀÚ¼¼ÇÑ ³»¿ëÀº ÇÏ´ÜÀÇ PDF ÂüÁ¶).
´ÙÄ«¶ó¹ÙÀÌ¿À¿¡¼ ÁøÇàÇÑ ½ÇÇèÀº °ø°³µÇ¾î ÀÖ´Â ÇÁ·ÎÅäÄÝ¿¡ µû¶úÀ¸¸ç, ¿ÜºÎ ±â°ü¿¡ ÀÇÇØ °ËÁõµÇ°Å³ª, ³»ºÎ R&DÆÀ¿¡¼ ÃÖÀûÈ°úÁ¤À» °ÅÄ¡Áö ¾Ê¾Ò½À´Ï´Ù. º» µ¥ÀÌÅÍ´Â ¿¹½ÃÀÇ ¸ñÀûÀ¸·Î¸¸ Á¦°øµÇ¸ç,
ÀÚ¼¼ÇÑ ÇÁ·ÎÅäÄÝÀº °Ô½ÃµÈ ¹®¼¸¦ Âü°í ¹Ù¶ø´Ï´Ù.
Áß±¹ Áúº´ÅëÁ¦¿¹¹æ¼¾ÅÍ¿¡¼´Â ´ç»ç Á¦Ç°À» ÀÌ¿ëÇÏ¿© COVID-19¸¦ °ËÃâÇÑ ³í¹®À» ¹ßÇ¥ÇÏ¿´´Ù (
Zhu et al. 2019).
COVID-19 ¿¬±¸¿¡ ÃÖÀûÀÎ ´ÙÄ«¶ó Real Time PCR Á¦Ç° ¶óÀÎ
COVID-19 È®»ê¹æÁö¸¦ À§ÇÑ ºü¸£°í Æí¸®ÇÑ Å¸¾× direct °Ë»ç¹ý
Ç¥ÁØ COVID-19 °Ë»ç´Â ºñ° ¸éºÀ °Ëü (nasal swab) ·ÎºÎÅÍ Á¤Á¦ÇÑ virus RNA¿¡¼ RT-qPCRÀ» ÀÌ¿ëÇÏ¿© SARS-CoV-2¸¦ °ËÃâÇÑ´Ù. ÀÌ·¯ÇÑ °Ë»ç¸¦ À§Çؼ´Â À̵éÀ» °¨¿°À¸·ÎºÎÅÍ º¸È£ÇÒ ¼ö ÀÖ´Â ¿ÂÀüÇÑ °³ÀÎ º¸È£ À屸 (PPE)°¡ ÇÊ¿äÇÒ »Ó¸¸ ¾Æ´Ï¶ó ¼÷·ÃµÈ »ùÇà äÃë Àü¹®°¡¿Í ±×¸®°í ºÒÆíÇÑ ºñ° ¸éºÀ Å×½ºÆ®°¡ ÇÊ¿äÇß´Ù. ÀÌ °úÁ¤Àº ÀÌ´Â »ùÇà äÃë°¡ ¾î·Á¿ï »Ó¸¸ ¾Æ´Ï¶ó, Å×½ºÆ® ºñ¿ëÀÌ ³ô°í, °Ë»ç ´çÀÏ °á°ú¸¦ Á¦°øÇÒ ¼ö ¾ø¾î ¿À·£ ½Ã°£ °á°ú¸¦ ±â´Ù·Á¾ß ÇÏ´Â µî COVID-19ÀÇ ´ëÀ¯Çà¿¡ ´ëÀÀÇϱ⿡´Â ¿©·¯ ÇѰ踦 °¡Áö°í ÀÖ´Ù.
ÀÌ·¯ÇÑ ¹®Á¦¸¦ °³¼±ÇÒ ¼ö ÀÖ´Â »õ·Î¿î Áø´Ü¹æ¹ý Áß Å¸¾× °Ë»ç´Â ¸î °¡Áö ÀåÁ¡ÀÌ ÀÖ´Ù. Ÿ¾× °Ë»ç´Â ºñħ½ÀÀûÀ̱⠶§¹®¿¡ ¼÷·ÃµÈ ±â¼úÀ» ¿äÇÏÁö ¾Ê°í, Áö¼ÓÀûÀ¸·Î ¸éºÀ ¹× Ư¼ö »ùÇà Ʃºê¿¡ ÀÇÁ¸ÇÏÁö ¾Ê¾Æµµ µÇ¸ç, Áõ»óÀÌ ½ÃÀ۵DZâ 1~3ÀÏÀü, ¾à 7ÀÏ ÈıîÁö °è¼ÓµÇ´Â Áúº´ÀÇ °¨¿°´Ü°è¿¡¼ È¿°úÀûÀ¸·Î °ËÃâµÉ ¼ö ÀÖ´Ù. ¹Ý¸é ºñ° ¸éºÀ »ùÇà °Ë»ç´Â Áõ»óÀÌ ¹ßÇöÇÑ ÈÄ¿¡ °¡Àå ½Å·ÚÇÒ ¼ö ÀÖ´Â Â÷ÀÌÁ¡µµ ÀÖ´Ù. (Wyllie
et al. 2020; Ott
et al. 2020).
Figure 1. Nasal swab testing for SARS-CoV-2 is lengthy and suffers from supply-chain issues that are preventing expansion efforts.
Á¤Á¦°¡ ÇÊ¿ä¾ø´Â SARS-Cov-2 °ËÃâ¹æ¹ýÀÇ °³¹ß Áö¿ø
ÆÒ´õ¹ÍÀ» ±Øº¹Çϱâ À§Çؼ´Â Àü¿ë ¸éºÀ°ú Ư¼ö »ùÇÃÆ©ºê°¡ ÇÊ¿ä¾ø´Â Àú·ÅÇÏ°í ½Å¼ÓÇÏ¸ç ºñħ½ÀÀûÀÎ ¹æ½ÄÀ» ÀÌ¿ëÇÑ ´ë±Ô¸ð Å×½ºÆ®·Î È®´ëÇÏ´Â °ÍÀÌ ÇÊ¿äÇÏ´Ù. ÀÌ·¯ÇÑ °Ë»ç´Â ½Å·ÚÇÒ ¼ö ÀÖ´Â ¹é½ÅÀ» Åõ¿©ÇÒ ¼ö ÀÖÀ» ¶§±îÁö ÀÏÁÖÀÏ¿¡ ¸î ¹ø¾¿ÀÌ¶óµµ ¹Ýº¹ÀûÀ¸·Î »ç¶÷µéÀÌ °Ë»ç¸¦ ÁøÇàÇÒ ¼ö ÀÖ°Ô ÇØÁÙ °ÍÀÌ´Ù.
ÃÖ±Ù, Ÿ¾×¿¡¼ RNA¸¦ Á¤Á¦ÇÏÁö ¾Ê°í ºü¸£°Ô Å×½ºÆ®ÇÏ´Â ¸î °¡Áö ÇÁ·ÎÅäÄÝÀÌ °³¹ßµÇ¾ú´Ù. ¸ê±Õ Æ©ºê¶ó¸é ¾î¶² °ÍÀÌµç »ùÇà äÃë¿¡ »ç¿ëÇÒ ¼ö ÀÖ°í, °£´ÜÇÑ ¹ÙÀÌ·¯½º RNA ÃßÃâ°úÁ¤À» °ÅÃÄ RT-qPCR¿¡ Á÷Á¢ »ç¿ëÇÏ°Ô µÈ´Ù. ÀÌ·¯ÇÑ ºü¸£°í °£´ÜÇÑ ÇÁ·ÎÅäÄÝÀº Àü¹® Àåºñ¸¦ ÇÊ¿ä·Î ÇÏÁö ¾Ê´Â ºñħ½ÀÀû »ùÇà äÃëÀÇ ÀåÁ¡ÀÌ ´õÇØÁ® ¸¹Àº Å×½ºÆ®¸¦ Çѹø¿¡ ÁøÇàÇÏ°íÀÚ ÇÏ´Â ³ë·ÂÀ» °¡¼ÓÈÇÏ°í ÀÖ´Ù.
½ÇÁ¦, ÇÙ»ê Á¤Á¦ ´Ü°è¸¦ ¿ìȸÇÏ´Â Extraction-free Ÿ¾× °Ë»ç´Â ´õ ºü¸£°í Àú·ÅÇÏ¸ç °ø±Þ¸Á À̽´¿Í °ü°è¾øÀÌ COVID-19 °Ë»ç¸¦ ÁøÇàÇÒ ¼ö ÀÖ°Ô ÇØÁÖ¾ú´Ù.
ÀÌ·¯ÇÑ Å¸¾×°Ë»ç´Â ºñ° ¸éºÀ »ùÇà °Ë»ç¿Í °ßÁÙ ¸¸Å ¹Î°¨Çϸç, ½ÇÁ¦ ¹Ì±¹ Àü¿ªÀÇ ¿©·¯ ´ëÇп¡¼ Á¤±âÀûÀ¸·Î Çлý, Á÷¿øµéÀ» °Ë»çÇÏ´Â µ¥ »ç¿ëµÇ°í ÀÖ´Ù.
Á¤Á¦ ¾ø´Â SARS-CoV-2 °ËÃâ
Ÿ¾×¿¡¼ º°µµÀÇ ¹ÙÀÌ·¯½º RNA Á¤Á¦ ¾øÀÌ ºü¸£°í Á¤È®ÇÏ°Ô SARS-CoV-2¸¦ °ËÃâÇÏ´Â ¹æ¹ýÀÌ ¸Å¿ì ÇÊ¿äÇÏ´Ù°í ¿©°ÜÁö´Â °¡¿îµ¥, ´ÙÄ«¶ó¿¡¼´Â ÀÌ·¯ÇÑ Crude sampleÀÇ gold standardÀÎ PrimeScript¢â III ¿ªÀü»çÈ¿¼Ò ±â¼úÀ» ÀÌ¿ëÇÏ¿© ÃÖÀûȵÈ
Direct One-Step RT-qPCR Mix for SARS-CoV-2 Á¦Ç°À» Ãâ½ÃÇÏ¿´´Ù.
RNAÀÇ Á¤Á¦°¡ ÇÊ¿ä¾ø´Â »õ·Î¿î °ËÃâ¹æ¹ýÀ» ¿¬±¸ÇÏ´Â ¿¬±¸ÀÚµéÀº º» Á¦Ç°ÀÇ °£´ÜÇÑ ÇÁ·ÎÅäÄÝ¿¡¼ ½ÃÀÛÇÒ ¼ö ÀÖ´Ù. Ÿ¾× »ùÇðú Àüó¸® ½Ã¾àÀ» È¥ÇÕ, ¿Ã³¸®ÇÑ ÈÄ, ÀÌ È¥ÇÕ¹°À» RT-qPCR master mix¿¡ ÷°¡ÇÏ¿© RT-qPCRÀ» ÇÑ´Ù. Àü󸮺ÎÅÍ °ËÃâ±îÁö Àüü ½ÇÇè°úÁ¤Àº 1½Ã°£ (Àüó¸® 5ºÐ, RT-qPCR ¾à 50ºÐ) ³»¿¡ ¿Ï·áµÇ±â¿¡ ºü¸¥ °á°ú µµÃâÀÌ °¡´ÉÇÏ´Ù. ÀÌ·¯ÇÑ °£´ÜÇÑ ¹æ¹ýÀ¸·Îµµ SARS-CoV-2¸¦ ³ôÀº ¹Î°¨µµ·Î °ËÃâÇÒ ¼ö ÀÖÀ½À» È®ÀÎÇÏ¿´´Ù (~SARS-CoV-2 RNA 10 copy °ËÃâ; Figure 2)
Figure 2. Direct One-Step PrimeScript RT-qPCR Mix uses a simple 3-step protocol to detect SARS-CoV-2 in saliva samples across a range of target RNA copy numbers. Panel A. Our simple protocol takes less than an hour to complete.
Panel B. Unpurified saliva samples spiked with synthetic RNA of the N1 and N2 regions of SARS-CoV-2 were processed according to the Direct One-Step PrimeScript RT-qPCR Mix for SARS-CoV-2 protocol, and positive detection was achieved for these targets as well as human RNase P (RP), the positive control.
COVID-19 ¿¬±¸¿¡ ÃÖÀûÀÎ ´ÙÄ«¶ó Real Time PCR Á¦Ç° ¶óÀÎ
[References]
Ott, I. M.,
et al. Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices.
MedRXiv. doi: 10.1101/2020.08.03.20165233 (2020).
Wyllie, A. L.
et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs.
MedRxiv. doi: https://doi.org/10.1101/2020.04.16.20067835 (2020).
hiPSC-based viral infection models
SARS-CoV-2´Â ÁÖ·Î »ç¶÷ÀÇ ±âµµ ³» »óÇǼ¼Æ÷¸¦ °¨¿°½ÃÅ°´Â °ÍÀ¸·Î ¾Ë·ÁÁ® ÀÖÀ¸³ª, ´Ù¾çÇÑ ¿¬±¸°á°ú¿¡¼ °£¼¼Æ÷ ¹× ÃéÀå ³»ºÐºñ ¼¼Æ÷, ½É±Ù¼¼Æ÷¿Í °°ÀÌ ACE2 receptor¸¦ ¹ßÇöÇÏ´Â ´Ù¸¥ À¯ÇüÀÇ ¼¼Æ÷¿¡µµ °¨¿° ´É·ÂÀÌ ÀÖ´Ù´Â °ÍÀÌ È®Àεǰí ÀÖ´Ù (Yang
et al. 2020). ÀÌ·¯ÇÑ COVID-19ÀÇ º´ÀαâÀüÀ» ÀÌÇØÇÏ°íÀÚ ÇÏ´Â ¿¬±¸ÀÚµéÀ» À§ÇØ ´ÙÄ«¶ó¹ÙÀÌ¿À´Â hiPSC À¯·¡ÀÇ ¼¼Æ÷ ¸ðµ¨À» Áö¿øÇÏ°í ÀÖ´Ù. hiPSC À¯·¡ÀÇ °£¼¼Æ÷, beta cell, ½É±Ù¼¼Æ÷, ¼ÒÀå»óÇǼ¼Æ÷¸¦ Æ÷ÇÔÇÏ´Â ¹è¾ç ½Ã½ºÅÛÀ¸·Î ¹ÙÀÌ·¯½º¿¡ °¨¿°µÇ°Å³ª ÀüÆĵǴ °úÁ¤À» È®ÀÎÇÔÀ¸·Î½á COVID-19 È®»êÀ» ¸·´Â »õ·Î¿î ¹æ¹ýÀ» ã´Âµ¥ µµ¿òÀÌ µÉ ¼ö ÀÖ´Ù. ´ÙÄ«¶ó¹ÙÀÌ¿ÀÀÇ hiPSC À¯·¡ ¼¼Æ÷µéÀº primary cell°ú À¯»çÇÑ ±â´É°ú Ư¼ºÀ» °¡Á® ½Å·Úµµ ³ô°í ÀçÇö¼º ÀÖ´Â °á°ú¸¦ ¾òÀ» ¼ö ÀÖÀ¸¸ç, COVID-19 ¿¬±¸¸¦ ½Å¼ÓÇÏ°Ô ÁøÇàÇÏ°íÀÚ ÇÏ´Â ¿¬±¸Àڵ鿡°Ô ÀÌ»óÀûÀÎ ¼¼Æ÷¸ðµ¨ÀÌ´Ù.
ADME-Tox studies
COVID-19 Ä¡·á¸¦ À§ÇÑ »õ·Î¿î ¾à¹° È常¦ ã¾Æ³»±â À§Çؼ´Â ¾à¹°ÀÇ Èí¼ö, ºÐÆ÷, ´ë»ç, ºÐºñ ¹× µ¶¼º (ADME-Tox)¸¦ Æò°¡ÇÒ ¼ö ÀÖ´Â ½Å·Úµµ ³ôÀº ¼¼Æ÷ ¸ðµ¨ÀÌ ÇÊ¿äÇÏ´Ù.
in vitro ½Ã½ºÅÛÀº
in vivo¿¡¼º¸´Ù »ýü ³» ¾à¹° ¾ÈÀü¼º°ú È¿À²À» º¸´Ù Á¤È®ÇÏ°Ô ¿¹ÃøÇÒ ¼ö ÀÖ¾î, ÀÓ»ó¿¡¼ ´õ ³ªÀº ¾à¹° È常¦ ÀÌ¿ëÇÒ ¼ö ÀÖ°Ô ÇÑ´Ù.
´ÙÄ«¶ó¹ÙÀÌ¿À¿¡¼ Á¦°øÇÏ´Â hiPSC À¯·¡ÀÇ °£¼¼Æ÷, ½É±Ù¼¼Æ÷´Â primary cell°ú À¯»çÇÑ Æ¯¼ºÀ» Áö³à, ¾à¹° Èĺ¸ÀÇ °£µ¶¼º°ú ½É±Ùµ¶¼ºÀ» Æò°¡ÇÒ ¼ö ÀÖ´Ù. °£Àº ü³» ¾à¹° ´ë»ç¿¡ ÁßÁ¡ÀûÀ¸·Î °ü¿©Çϱ⿡, hiPSC À¯·¡ °£¼¼Æ÷¸¦ ÀÌ¿ëÇϸé
in vitro »ó¿¡¼ ¾à¹° ´ë»ç¸¦ ¿¹ÃøÇÒ ¼ö ÀÖ´Ù.
Primary hepatocyte¸¦ ÀÌ¿ëÇÏ´Â °æ¿ì¿¡´Â,
Cellartis¢ç Power¢â Primary HEP MediumÀ¸·Î ¹è¾çÇϸé Àå±â°£ÀÇ ¾à¹° ´ë»ç °úÁ¤À» Á¤È®ÇÏ°Ô È®ÀÎÇÒ ¼ö ÀÖ´Ù.
°æ±¸ Åõ¿© ¾à¹°À» °³¹ßÁßÀÎ °æ¿ì¿¡´Â, hiPSC À¯·¡ÀÇ ¼ÒÀå»óÇǼ¼Æ÷ (hiPSC-derived intestinal epithelial cells)¸¦ ÀÌ¿ëÇØ ¾à¹° ±âÀÛ°ú Èí¼ö¸¦ Æò°¡ÇÒ ¼ö ÀÖ´Ù. ÀÌ ¼¼Æ÷´Â Çص¿ ÈÄ 5ÀÏ À̳» tight junctionÀ» Çü¼ºÇÒ »Ó ¾Æ´Ï¶ó, ¾à¹° ´ë»ç È¿¼Ò¿Í transporters¸¦ À¯ÀÇÇÏ°Ô ¹ßÇöÇÑ´Ù.
ÀÌ¿Í °°ÀÌ, hiPSC À¯·¡ ¼¼Æ÷ ¸ðµ¨À» »ç¿ëÇÏ¸é ¾à¹°ÀÌ »ýü ³»¿¡¼ ¾î¶»°Ô ÀÛ¿ëÇÒ Áö Á¤È®ÇÏ°Ô ¿¹ÃøÇÒ ¼ö ÀÖ¾î, ÀÓ»ó ½ÃÇè¿¡¼ÀÇ ¼º°ø °¡´É¼ºÀ» ³ôÀÏ ¼ö ÀÖ´Ù.
Various types of cell model derived from hiPSC
Hepatocytes
Cellartis¢ç enhanced hiPS-HEP v2 ¼¼Æ÷´Â 14ÀÏ °£ÀÇ assay window µ¿¾È ¼º¼÷ÇÑ °£ ¼¼Æ÷ Ư¼ºÀ» Áö¼ÓÀûÀ¸·Î ³ªÅ¸³¿À¸·Î½á °£¼¼Æ÷ ±â´É ¿¬±¸¿¡ »ç¿ëÇÒ ¼ö ÀÖ°í, ´ë»ç Áúȯ ¸ðµ¨¸µ, ½Å¾à °³¹ß, ¾à¹° ´ë»ç ¹× µ¶¼º ¿¬±¸¿¡ ÀÌ»óÀûÀÌ´Ù. ÀÌ ¼¼Æ÷´Â ¼º¼÷ÇÑ °£ ¼¼Æ÷¿¡¼ º¸ÀÌ´Â ÁÖ¿ä ¸¶Ä¿¸¦ ¹ßÇöÇÏ°í ÀÖÀ» »Ó ¾Æ´Ï¶ó albumin, urea ºÐºñ´É°ú glucose, lipidÀÇ Á¶Àý ±â´É µîÀÇ Æ¯¼ºÀ» º¸À¯ÇÏ°í ÀÖ´Ù. ¸¸¾à ¹ÙÀÌ·¯½º °¨¿°°ú Àü´Þ °úÁ¤¿¡¼ÀÇ À¯ÀüÀû ¿µÇâÀ» ¿¬±¸ÇÏ°íÀÚ ÇÑ´Ù¸é,
Cellartis¢ç iPS Cell to Hepatocyte Differentiation SystemÀ¸·Î ¿øÇÏ´Â hiPSC¿¡¼ hepatocyte¸¦ Á÷Á¢ ºÐȽÃų ¼ö ÀÖ´Ù.
Enhanced hiPS-HEP cells display markers of mature hepatocytes. RNA-seqÀ» ÅëÇØ, °£¼¼Æ÷ ÁÖ¿ä ¸¶Ä¿ÀÇ mRNA ¹ßÇöÀ» Á¤·® ºÐ¼®ÇÏ¿´´Ù.
(Panel A) 3Á¾ÀÇ hiPSC (C12, C18, C22)·ÎºÎÅÍ À¯·¡µÈ hiPS-HEP ¼¼Æ÷ (Çص¿ ÈÄ 12ÀÏ)¿Í primary hepatocytes (hphep cells, Çص¿ ÈÄ 1ÀÏ)¿¡¼
ALB°ú
¥á1AT ¹ßÇöÀ» ºñ±³ÇÏ¿´´Ù.
(Panel B) µ¿ÀÏÇÑ Á¶°Ç¿¡¼ °¢ ¼¼Æ÷ÁÖÀÇ Urea cycle enzyme mRNA ¹ßÇöÀ» È®ÀÎÇÏ¿´´Ù.
* ALB = albumin;
¥á1AT = alpha-1-antitrypsin;
CPS1 = carbamoyl-phosphate synthase;
OTC = ornithine carbamoyltransferase;
ASS1 = argininosuccinate synthase;
ASL = argininosuccinate lyase; and
ARG1 = arginase-1.
* Data are presented as mean values ¡¾ SEM.
Cardiomyocytes
Cellartis¢ç cardiomyocytes´Â hiPSC·ÎºÎÅÍ ¸Å¿ì ±ÕÀÏÇÑ ºÐÆ÷µµ¸¦ °®´Â ½É±Ù¼¼Æ÷·Î, »õ·Î¿î ¾à¹° Èĺ¸ ¹ß±¼, ¾à¸®ÇÐÀû ¾ÈÀü¼º, ½ÉÀå µ¶¼º Å×½ºÆ®¿Í °°Àº ºÐ¾ß¿¡ È°¿ëµÉ ¼ö ÀÖ´Ù. ÀÌ ¼¼Æ÷´Â Àΰ£ primary cardiomyocyte¿Í ¸Å¿ì À¯»çÇÑ Àü±â »ý¸®ÇÐÀû Ư¼ºÀ» º¸ÀδÙ. ¶ÇÇÑ, ½ÉÀåÀڱؿ¡ ¿¹»ó°ú °°Àº ¹ÝÀÀÀ» º¸ÀÓÀÌ È®ÀεǾú´Ù. ÇØ´ç ¼¼Æ÷´Â º°µµÀÇ ¼±º° °úÁ¤À̳ª À¯ÀüÀÚ Á¶ÀÛ °úÁ¤ ¾øÀ̵µ ¸Å¿ì È¿À²ÀûÀÎ ½É±Ù ºÐÈ ÇÁ·ÎÅäÄÝÀ» ÀÌ¿ëÇÏ¿© ºÐȵǾúÀ¸¸ç, Àΰ£ ½ÉÀå Á¶Á÷ °øÇÐÀ̳ª ´ë·®ÀÇ Àü±â »ý¸®ÇÐÀû ºÐ¼®¿¡µµ ¼º°øÀûÀ¸·Î ÀÌ¿ëµÇ°í ÀÖ´Ù.
Cellartis cardiomyocytes can be used to generate engineered heart tissue (EHT).
(Panel A) ½É±Ù ¼öÃà °úÁ¤ÀÇ EHT »çÁø
(Panel B) ½Ã°£ °æ°ú¿¡ µû¸¥ ½É±Ù ¼öÃà ´É·Â È®ÀÎ
Beta cells
Cellartis¢ç hiPS Beta Cells´Â ¹è¾Æ ¹ß´Þ°úÁ¤À» ¸ð¹æÇÏ¿© ÃÖÀûÈµÈ ºÐÈ ÇÁ·ÎÅäÄÝ·Î ºÐȵǾú°í, Àν¶¸° ºÐºñ ¹× Á¶Àý¿¡ °ü¿©ÇÏ´Â ÈÇÕ¹° ½ºÅ©¸®´×Àº ¹°·Ð GSIS ºÐ¼®, incretin ¹ÝÀÀ ¿¬±¸ µî¿¡ »ç¿ëµÇ°í ÀÖ´Ù. ´ÙÄ«¶ó¹ÙÀÌ¿À´Â °Ç°ÇÑ ±âÁõÀڷκÎÅÍ Á¦ÀÛÇÑ hiPSC À¯·¡ÀÇ ºÐÈ ¼¼Æ÷¿Í ´ç´¢º´ ¹Î°¨ÇÑ HLA À¯Çü (HLA-A * 02 : 01)À» °¡Áö´Â ±âÁõÀڷκÎÅÍ Á¦ÀÛÇÑ hiPSC À¯·¡ÀÇ ºÐÈ ¼¼Æ÷, ÃÑ µÎ Á¾ÀÇ Á¦Ç°À» Á¦°øÇÏ°í ÀÖ´Ù. µÎ Á¦Ç° ¸ðµÎ Beta cell ƯÀÌÀûÀÎ À¯ÀüÀÚ¿Í Àν¶¸°, C-peptide¸¦ ¹ßÇöÇÏ°í, Àν¶¸° °æ·Î¸¦ Ÿ°ÙÀ¸·Î ÇÏ´Â ¾à¹°¿¡¼µµ ÀûÀýÈ÷ ¹ÝÀÀÇÔÀ» È®ÀÎÇÏ¿´´Ù. µû¶ó¼, ¹ÙÀÌ·¯½º °¨¿°À¸·Î ÀÎÇÑ Beta cellÀÇ ¿¬±¸¿¡µµ È°¿ëµÇ±â¿¡ ÃæºÐÇÏ´Ù.
hiPS cell-derived beta cells consistently express key beta cell mRNAs. µÎ Á¾ÀÇ hiPSC·ÎºÎÅÍ ºÐÈÇÑ beta cellÀ» Çص¿ ÈÄ 14ÀÏ°£ ¹è¾çÇÑ ´ÙÀ½, primary islet°ú RNA-seqÀ» ÅëÇØ mRNA ¹ßÇöÀ» Á¤·®ÀûÀ¸·Î ºñ±³ ºÐ¼®ÇÏ¿´´Ù.
* PDX1 = pancreatic and duodenal homeobox 1; GCK = glucokinase; SLC2A1, SLC2A3 = Solute Carrier Family 2 Member 1 and 3; MAFB = MAF BZIP Transcription Factor B; NKX2.2 = NK2 homeobox 2; NKX6.1 = NK6 homeobox 1; and NEUROD1 = Neurogenic differentiation
* Data are presented as the mean values ¡¾ SEM.
Intestinal epithelial cells
Cellartis¢ç intestinal epithelial cells´Â hiPSC·ÎºÎÅÍ ºÐÈµÈ ¼ÒÀå»óÇǼ¼Æ÷·Î, ¼ÒÀå¿¡¼ ÀϾ´Â ¾à¹° Èí¼ö ¹× ´ë»ç¿Í ½Å¾à °³¹ß °úÁ¤¿¡¼ÀÇ ADME profiling, ¾à¹° Åõ°ú¼º ºÐ¼®¿¡ È°¿ëµÉ ¼ö ÀÖ´Ù. ÀÌ ¼¼Æ÷´Â ±â´ÉÀûÀÌ°í Åõ°ú °¡´ÉÇÑ À庮À» Çü¼ºÇϸç, villin°ú CDX2¸¦ ¹ßÇöÇÒ »Ó ¾Æ´Ï¶ó, Àΰ£ ´ëÀå¾Ï ¼¼Æ÷ÁÖÀÎ Caco-2¿¡ ºñÇØ ´ë»çÈ¿¼Ò CYP3A4 ¹× transporter PEPT1¿¡¼ ´õ ³ôÀº ¹ßÇöÀ» º¸ÀδÙ. µû¶ó¼,
Cellartis¢ç intestinal epithelial cellsÀ» ÀÌ¿ëÇÏ¸é ¾à¹° ´ë»ç Èí¼ö ¹× »óÈ£ÀÛ¿ë Æò°¡ °á°ú¸¦ ´õ Á¤È®ÇÏ°Ô ¿¹ÃøÇÒ ¼ö ÀÖ´Ù.
Cellartis intestinal epithelial cells recapitulate the microenvironment of the small intestine. Tight junction¿¡¼ ¹ßÇöÇÏ´Â ZO-1 ´Ü¹éÁúÀ» ¸é¿ª Çü±¤ ¿°»öÇßÀ» ¶§, Çص¿ ÈÄ 5Àϸ¸¿¡ ¼¼Æ÷ ³» tight junction (i.e., zonula occludens)À» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.
[ Drug discovery¿¡ ÃÖÀûÀÎ ´ÙÄ«¶ó Á¦Ç° ]
Vaccine development
SARS-CoV-2ÀÇ È®»êÀ¸·Î virus¿¡ ´ëÇÑ ¸é¿ª ¹ÝÀÀÀ» À¯µµÇÒ ¼ö ÀÖ´Â epitope¸¦ È®ÀÎÇϰųª, »õ·Î¿î ¹é½ÅÀ» °³¹ßÇÏ´Â µîÀÇ ¿¬±¸°¡ ºü¸¥ ¼Óµµ·Î ÁøÇàµÇ°í ÀÖ´Ù.
Takara Bio´Â ÀÌ·¯ÇÑ ¿¬±¸¸¦ Áö¿øÇϱâ À§ÇØ ¹é½Å °³¹ß °úÁ¤¿¡¼ÀÇ ´Ù¾çÇÑ Á¦Ç°À» Á¦°øÇÏ°í ÀÖ´Ù.
[ ¹é½Å °³¹ß workflow ]
Viral and host genomics
SARS-CoV-2 virusÀÇ ¸é¿ª ¹ÝÀÀÀ» À¯¹ßÇÏ´Â ´Ù¾çÇÑ Ç׿ø epitope¸¦ È®ÀÎÇϱâ À§ÇØ, whole-genome sequencing (WGS)Àº ÇʼöÀûÀÌ´Ù. ´ç»ç´Â SARS-CoV-2ÀÇ full-length viral genome ¼¿À» ºÐ¼®Çϱâ À§ÇØ
SMARTer¢ç Stranded Pico v2 kit¸¦ Àû¿ëÇÑ »ç·Ê¸¦ º¸À¯ÇÏ°í ÀÖ´Ù.
COVID-19¿¡ °¨¿°µÇ¾ú°Å³ª ȸº¹ÇÑ »ç¶÷À¸·ÎºÎÅÍ RNA-seqÀ» ÀÌ¿ëÇÑ immune profilingÀ» ÁøÇàÇϸé, Ç׿ø°ú ¹é½Å Ÿ°Ù¿¡ ´ëÇÑ Ãß°¡ÀûÀÎ Á¤º¸¸¦ ¾òÀ» ¼ö ÀÖ´Ù. ´ç»çÀÇ °í°¨µµ
SMARTer¢ç Human BCR and TCR profiling kits¸¦ ÀÌ¿ëÇϸé Áúº´ °¨¿°ºÎÅÍ È¸º¹±îÁö
Áúº´ÀÌ ÁøÇàµÇ´Â µ¿¾ÈÀÇ BCR (B-cell receptor)°ú TCR (T-cell receptor) clonotype repertories¿¡ ´ëÇÑ º¯È¸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
Clone vaccine targets
High-throughput cloningÀÌ °¡´ÉÇÑ In-Fusion
¢ç HD Cloning ±â¼úÀ» ÀÌ¿ëÇϸé, viral receptor¿Í ¹é½Å construct¸¦ ºü¸£°Ô ±¸ÃàÇÒ ¼ö ÀÖ´Ù.
*Highly efficient |
0.5 ~ 15 kb »çÀÌÀÇ insert Å©±â¿¡¼ 95% ÀÌ»óÀÇ cloning È¿À² |
*Sequence independent |
Any insert, Any vector, Any locus cloning °¡´É |
*Seamless construction |
ºÒÇÊ¿äÇÑ ¿°±â Ãß°¡ ¾øÀ½ |
*Versatile |
ÇϳªÀÇ Á¦Ç°À¸·Î Multiple insert cloningºÎÅÍ site-directed mutagenesis±îÁö °¡´É |
[ In-Fusion¢ç HD Cloning technologyÀ» ÀÌ¿ëÇÑ SARS-CoV-2 ¿¬±¸ ³í¹® ]
-
Kato, H. et al. Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice. PLoS One. 14, DOI:10.1371/journal.pone.0223684 (2019).
-
Letko, L., Marzi, A., & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 6, 562-569 (2020).
-
Terada, Y., Kawachi, K., Matsuura, Y., Wataru, W., Kamitani, W. MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA. Virol. 511, 95-105 (2019).
Express and purify vaccine targets
´ç»çÀÇ
BacPAK¢â Baculovirus Expression SystemÀ» ÀÌ¿ëÇÏ¸é ¹é½Å °³¹ßÀ» À§ÇÑ Èĺ¸ ´Ü¹éÁúÀ» »ý»êÇÒ ¼ö ÀÖÀ¸¸ç, ½±°Ô scale upÀÌ °¡´ÉÇÏ´Ù. ÀÌ ½Ã½ºÅÛÀ» ÀÌ¿ëÇϸé ÀÚ¿¬ÀûÀ¸·Î ¹ßÇöµÇ´Â ´Ü¹éÁú°ú ±¸Á¶, »ý¹°ÇÐÀû È°¼º, ¸é¿ª ¹ÝÀÀ¼º¿¡ ÀÖ¾î ¸Å¿ì À¯»çÇÑ Å¸°Ù ´Ü¹éÁúÀ» »ý»êÇÒ ¼ö ÀÖ´Ù. His-tagÀ» Æ÷ÇÔÇÏ´Â ´Ü¹éÁúÀº
Capturem¢â His-Tagged Purification kit·Î ½±°Ô Á¤Á¦ÇÒ ¼ö ÀÖ´Ù.
*Fast, convenient workflow |
spin column/plate/filtration device¸¦ ÀÌ¿ëÇØ »ó¿Â¿¡¼ 5~15ºÐ ³» Æí¸®ÇÏ°Ô ´Ü¹éÁú Á¤Á¦ |
*High purity |
ÀÛÀº bed volumeÀ¸·Î ¿À¿°¹° È¥ÀÔ ÀûÀ½ |
*Compatible with a wide range of additives |
EDTA, DTT, BME, glycerol, TCEP µî ´Ù¾çÇÑ Ã·°¡Á¦¿Í ȣȯµÇ¾î, ¹öÆÛ ±³Ã¼ ¾øÀÌ »ç¿ë |
*Versatile |
Mammalian/Bacterial cell lysate¿Í ¼¼Æ÷ ¹è¾ç »óÃþ¾×¿¡¼ ½±°Ô Á¤Á¦ °¡´É |
[ Capturem His technologyÀ» ÀÌ¿ëÇÑ SARS-CoV-2 ¿¬±¸ ³í¹® ]
-
Do, V. T. et al. Recombinant adenovirus carrying a core neutralizing epitope of porcine epidemic diarrhea virus and heat-labile enterotoxin B of Escherichia coli as a mucosal vaccine. Arch. Virol. 165, 609-618 (2020).
-
Martinez-Hernandez, S. L. et al. An anti-amoebic vaccine: generation of the recombinant antigen LC3 from Entamoeba histolytica linked to mutated exotoxin A (PE¥ÄIII) via the Pichia pastoris system. Biotechnol. Lett. 39, 1149-1157 (2017).
Immunize mice with vaccine targets, screen sera for antiviral activity and optimize promising candidates
´ç»çÀÇ °í°¨µµ
SMARTer¢ç Mouse BCR and TCR profiling kits¸¦ ÀÌ¿ëÇϸé Áúº´ °¨¿°ºÎÅÍ È¸º¹±îÁö Áúº´ÀÌ ÁøÇàµÇ´Â µ¿¾ÈÀÇ BCR (B-cell receptor)°ú TCR (T-cell receptor) clonotype repertories¿¡ ´ëÇÑ º¯È¸¦ ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.°¡´É¼º ÀÖ´Â ¹é½Å Èĺ¸¹°ÁúµéÀº µ¿¹°¸ðµ¨¿¡¼ ¹ÙÀÌ·¯½º ³ëÃâ ÀüÈÄ¿¡¼ÀÇ Ç×ü, T ¼¼Æ÷ º¯È¸¦ ºñ±³, ºÐ¼®ÇÔÀ¸·Î½á ÃÖÀûÈ °úÁ¤À» ÁøÇàÇÒ ¼ö ÀÖ´Ù.
Assess effectiveness of selected final vaccine candidates in clinical trials
¸é¿ª ¹ÝÀÀÀ» ¸ð´ÏÅ͸µ Çϱâ À§Çؼ´Â ÃÖÁ¾ ¹é½Å Èĺ¸ ¹°ÁúÀÇ È¿´ÉÀ» Æò°¡ÇØ¾ß ÇÑ´Ù. NGS¸¦ ÀÌ¿ëÇÑ BCR/TCR repertoire profilingÀ» ÅëÇØ, clonotypeÀÇ º¯È¸¦ Á¤È®ÇÏ°Ô È®ÀÎÇÒ ¼ö ÀÖ´Ù.
[ COVID-19 vaccine development¿¡ ÃÖÀûÀÎ ´ÙÄ«¶ó Á¦Ç° ]
½ÅÁ¾ Äڷγª ¹ÙÀÌ·¯½º (SARS-CoV-2)´Â ƯÁ¤ ȯÀÚ¿¡°Ô¼ »çÀÌÅäÄ«ÀÎ Æødz ÁõÈıº ¹× ±Þ¼º È£Èí °ï¶õ ÁõÈıº (ARDS)À» Æ÷ÇÔÇÑ ½É°¢ÇÑ Áúº´À» À¯¹ßÇÏ¿© Á¾Á¾ »ç¸Á¿¡ À̸£°Ô ÇÑ´Ù. ÇöÀç±îÁö´Â COVID-19ÀÇ È¿°úÀûÀÎ Ä¡·áÁ¦°¡ °³¹ßµÇÁö ¾Ê¾ÒÀ¸³ª, Áß°£¿± Áٱ⼼Æ÷ (MSC)ÀÇ ¸é¿ª Á¶Àý È¿°ú¸¦ ÀÌ¿ëÇÑ »õ·Î¿î Ä¡·áÁ¦·Î ¶°¿À¸£°í ÀÖ´Ù. ÀÌ·¯ÇÑ ¸é¿ª Á¶Àý È¿°ú´Â EV (e.g. exosome)¿Í °°Àº ºÐºñ¹°ÁúÀ» ÅëÇØ »çÀÌÅäÄ«ÀÎ Æødz ÁõÈıº ¹× ARDS ¹ß»ýÀ» °¨¼Ò½ÃÅ´À¸·Î½á ³ªÅ¸³ª´Â °ÍÀ¸·Î º¸ÀδÙ. ¶ÇÇÑ, MSC´Â Æó ¼¶À¯ÁõÀ» ¿ÏȽÃÅ°°í ¼Õ»óµÈ Á¶Á÷À» º¹±¸ÇÒ ¼ö ÀÖ¾î, ½ÇÁ¦·Î COVID-19 °¨¿°À¸·Î ÀÎÇØ ¹ß»ýÇϴ ȣÈí °ï¶õ µî ÁßÁõ Æó Áúȯ¿¡ MSC¿Í ºÐºñ¹°ÁúÀ» È°¿ëÇÏ´Â Ãʱ⠿¬±¸°¡ ÁøÇàµÇ°í ÀÖ´Ù (Leng
et al. 2020; Sengupta
et al. 2020).
Cellartis MSC Xeno-Free Culture Medium
- ¾ÈÁ¤ÀûÀΠǥÇöÇü (CD marker ¹ßÇö, ´ÙºÐÈ´É) À¯Áö·Î, ÃÖÀûÀÇ ¼¼Æ÷ ¸ðµ¨ ½Ã½ºÅÛ Á¦°ø
- °·ÂÇÑ È®´ë ¹è¾ç ´É·Â (ÃÖ´ë 100x / week)À¸·Î ¼¼Æ÷ Ä¡·áÁ¦ ¿¬±¸¿¡ È°¿ëÇϱ⿡ ÃæºÐÇÑ ¾çÀÇ ¼¼Æ÷ ȹµæ
- Coating material ¾øÀÌ ¹è¾çÇÒ ¼ö ÀÖ¾î, À¯¿¬¼º°ú »ç¿ë ¿ëÀ̼º Áõ´ë
Capturem Extracellular Vesicle Isolation Kits
- MiniprepÀÇ °æ¿ì ÃÖ´ë 850 §¡, MaxiprepÀÇ °æ¿ì ÃÖ´ë 24 §¢ÀÇ »ýü »ùÇ÷κÎÅÍ 30ºÐ À̳»¿¡ °í¼øµµ Extracellular vesicle (EV) Á¤Á¦
- ÈÄ¼Ó ½ÇÇè Àû¿ë¿¡ ÃæºÐÇÑ ¾çÀÇ EV Á¤Á¦ °¡´É
(MiniprepÀÇ °æ¿ì ÃÖ´ë 1010 yield, MaxiprepÀÇ °æ¿ì ÃÖ´ë 1011 yield)
- Á¤Á¦µÈ EV¿¡¼ exosome ´Ü¹éÁú ¸¶Ä¿ ¹ßÇö È®ÀÎ
- AlbuminÀ̳ª calnexin°ú °°Àº ¿À¿° ¹°Áú È¥ÀÔÀÌ ±Øµµ·Î ÀûÀ½
- ÇϳªÀÇ Á¦Ç°À¸·Î ´Ù¾çÇÑ »ùÇà (Ç÷Àå, Ç÷û, ¼Òº¯, ¿ìÀ¯, Ÿ¾×, ¼¼Æ÷ Á¶Àý ¹èÁö ¹× ³úô¼ö¾× (CSF))¿¡ Àû¿ë °¡´É
[ Therapeutics research¿¡ ÃÖÀûÀÎ ´ÙÄ«¶ó Á¦Ç° ]
[ References ]
- Leng, Z.
et al. Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia.
Aging and Disease 11, 216-228 (2020).
- Senugupta, V.
et al. Exosomes Derived from Bone Marrow Mesenchymal Stem Cells as Treatment for Severe COVID-19.
Stem Cells Dev. May 12, 2020. doi: 10.1089/scd.2020.0080. Online ahead of print.
Why do we need pseudotyped SARS-CoV-2
SARS-CoV-2°¡ À¯ÇàÇϸé¼, ¹é½ÅÀ̳ª Ä¡·áÁ¦ °³¹ß ¿¬±¸¿¡ °ü½ÉÀÌ ÁýÁߵǰí ÀÖ´Ù. ÇÏÁö¸¸, ¹ÙÀÌ·¯½º ÀÚüÀÇ °¨¿° À§Ç輺, ÀüÆÄ·Â µîÀ¸·Î ÀÎÇØ BSL-3 ½Ã¼³¿¡ ±¹ÇÑµÇ¾î ¿¬±¸¸¦ ÁøÇàÇÒ ¼ö Àֱ⿡, ´Ù¼öÀÇ ¿¬±¸ ±â°ü¿¡¼´Â Ÿ ¹ÙÀÌ·¯½ºÀÇ backbone¿¡¼ SARS-CoV-2ÀÇ spike proteinÀ» ¹ßÇöÇÏ´Â pseudotyped virus¸¦ °³¹ßÇÏ¿© ÀÌ¿¡ ´ëÇÑ È¿¿ë¼ºÀ» È®ÀÎÇÏ¿´´Ù. À̸¦ ÅëÇØ, º¸´Ù ´õ ¾ÈÀüÇÏ°Ô, BSL-2 ½Ã¼³¿¡¼µµ SARS-CoV-2 ¿¬±¸¸¦ ÁøÇàÇÒ ¼ö ÀÖ´Ù (Ni
et al. 2020, Shang
et al. 2020).
How to make pseudotyped SARS-CoV-2
´ÙÄ«¶ó¹ÙÀÌ¿ÀÀÇ Lentivirus packaging ±â¼úÀ» Àû¿ëÇÑ
Lenti-X¢â SARS-CoV-2 Packaging Single Shots¸¦ ÀÌ¿ëÇϸé, SARS-CoV-2 spike-pseudotypedÀ» °¡Áö´Â °í¿ª°¡ÀÇ Lentivirus¸¦ ¼Õ½±°Ô »ý»êÇÒ ¼ö ÀÖ´Ù. ÀÌ Á¦Ç°Àº spike proteinÀ» Æ÷ÇÔÇÏ´Â packaging plasmid¿Í transfection ½Ã¾àÀÌ ÃÖÀûÀÇ ºñÀ²·Î È¥ÇյǾî, µ¿°á°ÇÁ¶ ÇüÅ·ΠÁ¦°øµÈ´Ù. µû¶ó¼, Lentivirus¸¦ óÀ½ Á¦ÀÛÇÏ´Â ¿¬±¸ÀÚ¶óµµ º°µµÀÇ ÃÖÀûÈ °úÁ¤À̳ª ½ÃÇàÂø¿À ¾øÀÌ 2~3ÀÏÀÌ¸é ¹ÙÀÌ·¯½º¸¦ ¾òÀ» ¼ö ÀÖ´Ù.
º» Á¦Ç°Àº spike protein variant¿¡ µû¶ó Á¦Ç°À» ¼±ÅÃÇÒ ¼ö ÀÖ´Ù.
-> WT (full length) - Wuhan-Hu-1 (NC_045512.2)·ÎºÎÅÍ À¯·¡ÇÑ SARS-CoV-2 ¼¿À» ÀÌ¿ëÇßÀ¸¸ç, mammalian cell¿¡¼ ¹ßÇöµÉ ¼ö ÀÖµµ·Ï ÃÖÀûÈ.
-> D614G (full length) - ÇöÀç À¯ÇàÇÏ´Â ´ëºÎºÐÀÇ ¹ÙÀÌ·¯½º¿¡¼ º¸ÀÌ´Â D614G mutation spike protein coding sequence Æ÷ÇÔ (Isabel et al. 2020).
-> B.1.351 (full length) - South Africa·ÎºÎÅÍ À¯·¡µÈ °ÍÀ¸·Î È®ÀεǴ B.1.351 mutation spike protein coding sequence Æ÷ÇÔ (Johnson et al. 2020).
-> WT (truncated) / D614G (truncated) / B.1.351 (truncated) - WT spike¿Í D614G, B.1.351 spike coding sequence¿¡¼ C-terminiÀÇ ER retention signalÀ» Á¦°ÅÇÔÀ¸·Î½á ¹ÙÀÌ·¯½º°¡ ¹ßÇöÇÏ´Â spike proteinÀ» Áõ°¡½ÃÄÑ, ³ôÀº °¨¿°·Â È®ÀÎ (Johnson
et al. 2020)
When could we use pseudotyped SARS-CoV-2
SARS-CoV-2ÀÇ ¹é½Å°ú Ä¡·áÁ¦ °³¹ßÀ» À§Çؼ´Â ÀϹÝÀûÀ¸·Î neutralization ´É·ÂÀ» È®ÀÎÇÑ´Ù. SARS-CoV-2 spike proteinÀº ¼÷ÁÖÀÇ ACE2 receptor¿Í °áÇÕÇÏ¿© ¼¼Æ÷¸¦ °¨¿°½ÃÅ°´Âµ¥, Èĺ¸ ¹é½Å°ú Ä¡·áÁ¦´Â ´ëºÎºÐ spike protein°ú ACE2 receptorÀÇ °áÇÕÀ» ¹æÇØÇÏ´Â ±âÀüÀ¸·Î ¼³°èµÇ°í ÀÖ´Ù.
Lenti-X¢â SARS-CoV-2 Packaging Single ShotsÀ¸·Î »ý¼ºµÈ ¹ÙÀÌ·¯½º°¡ µµÀÔµÈ ¼¼Æ÷´Â ¸®Æ÷ÅÍ·Î luciferase³ª ZsGreen1À» ¹ßÇöÇÏ°Ô µÇ°í, Èĺ¸ ¹°ÁúÀ» ó¸®ÇßÀ» ¶§ ÀÌ ¸®Æ÷ÅÍÀÇ ¹ßÇö Â÷À̸¦ È®ÀÎÇÏ¿©, ¹ÙÀÌ·¯½º °¨¿° ¾ïÁ¦, ¹æÁö ¼º´ÉÀ» Æò°¡ÇÏ´Â µ¥ È°¿ëÇÒ ¼ö ÀÖ´Ù.
Human ACE2 293T Cell Line (Code 631289)´Â receptor proteinÀÎ ACE2¸¦ ³ô°Ô ¹ßÇöÇϵµ·Ï Á¦ÀÛÇÑ 293T cell lineÀ¸·Î, neutralization assay¿¡ ÃÖÀûȵǾî ÀÖ´Ù. µû¶ó¼, ¹ÙÀÌ·¯½º¿Í receptor °£ÀÇ »óÈ£ÀÛ¿ëÀ̳ª ¹ÙÀÌ·¯½º °¨¿° °æ·Î µîÀÇ ¿¬±¸¿¡ È°¿ëÇÒ ¼ö ÀÖ´Ù.
[ ÇÔ²² »ç¿ë °¡´ÉÇÑ Á¦Ç° ]
[ References ]
- Ni, L.
et al. Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals.
Immunity.
52, 971-977 (2020).
- Shang, J.
et al. Cell entry mechanisms of SARS-CoV-2.
Proc Natl Acad Sci USA.
117, 11727-11734 (2020).
- Isabel, S
. et al. Evolutionary and structural analyses of SARS-CoV-2 D614G spike protein mutation now documented worldwide.
Sci Rep.
10, 14031 (2020).
- Madhi, S
. et al. Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant.
N Engl J Med. 384, 1885-1898 (2021).
- Johnson, MC.
et al. Optimized Pseudotyping Conditions for the SARS-COV-2 Spike Glycoprotein.
J Virol.
94, e01062-20 (2020).
Next-Generation Sequencing (NGS)´Â ȯ°æ, µ¿¹°, »ç¶÷À¸·ÎºÎÅÍ ¹ÙÀÌ·¯½º¸¦ °ËÃâÇÏ°í ±× Æ¯¼ºÀ» È®ÀÎÇÒ ¼ö ÀÖ´Â À¯¿ëÇÑ µµ±¸·Î »ç¿ëÇÒ ¼ö ÀÖ´Ù
(Kustin
et al. 2019).
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Monitoring of the environment
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Takara BioÀÇ SMARTer RNA-seq Á¦Ç°µéÀº ÀûÀº ¾çÀÇ RNA·ÎºÎÅÍ ¹Î°¨µµ ÀÖ´Â °ËÃâÀÌ °¡´ÉÇÒ »Ó ´õ·¯ random primingÀ» ÀÌ¿ëÇÑ ¿ªÀü»ç¹ÝÀÀÀ» ÅëÇØ ¼Õ»ó Á¤µµ°¡ ´Ù¾çÇÑ RNA¿¡µµ Àû¿ëÇÒ ¼ö ÀÖ´Ù. ƯÈ÷,
SMARTer¢ç Pico v2 RNA-seq ¶Ç´Â
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References
Hryhorowicz, Szymon, et al. "European context of the diversity and phylogenetic position of SARS-CoV-2 sequences from Polish COVID-19 patients." Journal of applied genetics 62.2 (2021): 327-337.
Thair, Simone A., et al. "Transcriptomic similarities and differences in host response between SARS-CoV-2 and other viral infections." IScience 24.1 (2021): 101947.
Ladner, Jason T., et al. "Defining the Pandemic at the State Level: Sequence-Based Epidemiology of the SARS-CoV-2 virus by the Arizona COVID-19 Genomics Union (ACGU)." medRxiv (2020).
Pfefferle, Susanne, et al. "Complete genome sequence of a SARS-CoV-2 strain isolated in Northern Germany." Microbiology resource announcements 9.23 (2020): e00520-20.
Lu, Jing, et al. "Genomic epidemiology of SARS-CoV-2 in Guangdong province, China." Cell 181.5 (2020): 997-1003.
Chen, Long, and Li Zhong. "Genomics functional analysis and drug screening of SARS-CoV-2." Genes & diseases 7.4 (2020): 542-550.
Perez-Bermejo, Juan A., et al. "SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients." BioRxiv (2020).
Kustin, Talia, et al. "A method to identify respiratory virus infections in clinical samples using next-generation sequencing."
Scientific reports 9.1 (2019): 1-8.
Understanding the effects of viral infection
Takara BioÀÇ
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SMART-Seq v4Á¦Ç°Àº ¹ÙÀÌ·¯½ºÀÇ °¨¿°°ú Ä¡·á °úÁ¤¿¡¼ÀÇ
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References
Rodda, Lauren B., et al. "Functional SARS-CoV-2-specific immune memory persists after mild COVID-19." Cell 184.1 (2021): 169-183.
Hoang, Timothy N., et al. "Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques." Cell 184.2 (2021): 460-475.
Jackson, Daniel J., et al. "Association of respiratory allergy, asthma, and expression of the SARS-CoV-2 receptor ACE2." Journal of Allergy and Clinical Immunology 146.1 (2020): 203-206.
Stanley, Kate E., et al. "Coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues." Fertility and sterility 114.1 (2020): 33-43.
Kase, Yoshitaka, and Hideyuki Okano. "Expression of ACE2 and a viral virulence-regulating factor CCN family member 1 in human iPSC-derived neural cells: implications for COVID-19-related CNS disorders." Inflammation and regeneration 40.1 (2020): 1-8.
Sharma, Arun, et al. "Human iPSC-derived cardiomyocytes are susceptible to SARS-CoV-2 infection." Cell Reports Medicine 1.4 (2020): 100052.
Giobbe, Giovanni Giuseppe, et al. "SARS-CoV-2 infection and replication in human fetal and pediatric gastric organoids." bioRxiv (2020).
Monitoring the immune response
¹ÙÀÌ·¯½º¿¡ °¨¿°µÇ¾úÀ» ¶§¿Í Ä¡·á °úÁ¤¿¡¼ÀÇ ¸é¿ª ¹ÝÀÀÀ» ÀÌÇØÇϱâ À§Çؼ´Â Àü»çüÀÇ profiling º¯È °úÁ¤À» ÀÌÇØÇÏ´Â °ÍÀÌ Áß¿äÇÏ´Ù. ¿¹·Î, COVID-19¿¡ °¨¿°µÇ¾úÀ» ¶§ B cell°ú T cellÀ¸·ÎºÎÅÍ
Clonotype¿¡ ¾î¶² º¯È°¡ ¹ß»ýÇß´ÂÁö, ÇöÀç »óÅÂ¿Í °¨¿° ÈÄ clontype ´Ù¾ç¼º¿¡ ¹ÌÄ£ ¿µÇâÀÌ ÀÖ´ÂÁö, Ä¡·á¸¦ ÅëÇØ ¾î¶°ÇÑ ¾ç»óÀ» º¸ÀÌ´ÂÁö µî, °¨¿°À¸·Î ÀÎÇÑ ¿µÇâÀ» È®ÀÎÇغ¼ ¼ö ÀÖ´Ù.
Takara Bio´Â ÀÌ·¯ÇÑ ¿µÇâÀ» ºÐ¼®ÇÒ ¼ö ÀÖµµ·Ï human
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References
Kustin, T.
et al. A method to identify respiratory virus infections in clinical samples using next-generation sequencing.
Sci. Rep. 9, 1-8 (2019).
Wu, F
. et al. A new coronavirus associated with human respiratory disease in China.
Nature 579, 265-269 (2020).