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¹ü¿ë¼º No.1, Plasmid DNA¿Í siRNA/miRNA ¸ðµÎ Àû¿ë

TransIT-X2¢ç Transfection Reagent

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
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Mirus
MIR 6003
TransIT-X2 Dynamic Delivery System
°ü·ÃÇмú ±¸¸ÅÇϱâ
0.3 ml
°¡°Ý¹®ÀÇ   ml061-transit-x2-dynamic-delivery-system-user-protocol.pdf, TranIT-X2_protocol_optimization_card.pdf
Mirus
MIR 6004
TransIT-X2 Dynamic Delivery System
°ü·ÃÇмú ±¸¸ÅÇϱâ
0.75 ml
°¡°Ý¹®ÀÇ  
Mirus
MIR 6000
TransIT-X2 Dynamic Delivery System
°ü·ÃÇмú ±¸¸ÅÇϱ⠻ùÇýÅû 
1.5 ml
°¡°Ý¹®ÀÇ  
Mirus
MIR 6005
TransIT-X2 Dynamic Delivery System
°ü·ÃÇмú ±¸¸ÅÇϱâ
1.5 ml¡¿5
°¡°Ý¹®ÀÇ  
Mirus
MIR 6006
TransIT-X2 Dynamic Delivery System
°ü·ÃÇмú ±¸¸ÅÇϱâ
1.5 ml¡¿10
°¡°Ý¹®ÀÇ  



  • ´Ù¾çÇÑ cell type°ú ´Ù¾çÇÑ application¿¡ °íÈ¿À² Àû¿ë °¡´É (±×¸² 1)
  • DNA¿Í siRNA/miRNA, co-transfection ¸ðµÎ °¡´É
  • Primary cellÀ» Æ÷ÇÔÇÑ ´Ù¾çÇÑ ¼¼Æ÷¿¡ È¿À²ÀûÀ¸·Î µµÀÔ: ÁÖ¿ä cell lineº° ½ÇÇè °¡À̵å¶óÀÎ º¸±â (Ŭ¸¯)
  • Animal origin Free
  • CRISPR/Cas9 ½ÇÇè¿¡µµ ÃÖÀû: Cas9-sgRNA Á¶ÇÕ¿¡ µû¸¥ ½ÇÇè °¡À̵å¶óÀÎ Á¦°ø
    TransIT-X2¢ç for CRISPR RNP Delivery (PDF ¸µÅ©)
    TransIT-X2¢ç for CRISPR RNP + ssODN Delivery Protocol (PDF ¸µÅ©)
    TransIT-X2¢ç for CRISPR pDNA/gRNA Delivery Protocol (PDF ¸µÅ©)
    TransIT-X2¢ç for CRISPR Plasmid Delivery Protocol (PDF ¸µÅ©)

±×¸² 1. TransIT¢ç-X2 SystemÀº ´Ù¾çÇÑ cell type¿¡¼­ °íÈ¿À²·Î À¯ÀüÀÚ ¹ßÇö °¡´É. TransIT-X2¢ç Dynamic Delivery System°ú Lipofectamine¢ç 2000 Transfection Reagent¸¦ »ç¿ëÇؼ­ luciferase¸¦ encodingÇÏ´Â plasmid DNA¸¦ 41°³ÀÇ ´Ù¸¥ cell type¿¡ 3 : 1 (reagent : DNA)ÀÇ ºñÀ²·Î transfection ÇÏ¿´´Ù. Transfection 24½Ã°£ ÈÄ¿¡ standard luciferase assay¸¦ ÁøÇàÇÑ °á°ú TransIT-X2¢ç¸¦ »ç¿ëÇÏ´Â °æ¿ì 41°³ Áß 36°³ÀÇ cell type¿¡¼­ luciferase È°¼ºÀÌ µ¿µîÇϰųª ´õ ³ô¾Ò´Ù. ÀÌ Áß 17°³ÀÇ cell type¿¡¼­ TransIT-X2¢ç °¡ 2¹è ÀÌ»ó ³ô¾Ò°í ÇØ´ç cell typeÀ» ¢Ô·Î Ç¥½ÃÇÏ¿´´Ù.
Á¦Ç° ¼³¸í
TransIT-X2¢ç Dynamic Delivery SystemÀº non-liposomal polymeric ½Ã½ºÅÛÀ¸·Î primary cellÀ» Æ÷ÇÔÇÑ ´Ù¾çÇÑ cell type¿¡¼­ °íÈ¿À² transfectionÀÌ °¡´ÉÇÏ´Ù. ÀÌ Á¦Ç°À» »ç¿ëÇϸé DNA, siRNA/RNAi ¹× DNA¿Í siRNA µîÀÇ co-transfectionÀ» ÁøÇàÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ serum compatible transfectionÀÌ Æ¯Â¡À¸·Î transfection ÈÄ º°µµÀÇ media change°¡ ÇÊ¿äÇÏÁö ¾Ê´Ù´Â ÀåÁ¡ÀÌ ÀÖ´Ù.
º¸Á¸ -20¡É
°ü·ÃÁ¦Ç°
- Á¦ÇÑÈ¿¼Ò site¸¦ °í·ÁÇÏÁö ¾Ê´Â PCR cloing ¿øÁ¶ In-Fusion¢ç Snap Assembly Master Mix (Code 638947)
- Mycoplasma ¿À¿° È®ÀÎ ½Ã¾à
   : Nested PCR·Î Á¤¹Ð °ËÃâ TaKaRa PCR Mycoplasma Detection Set
   : °í°¨µµÀÇ cycling probe qPCR ÀÌ¿ë CycleavePCR Mycoplasma Detection Kit
   : Luminometer·Î 20ºÐ¸¸¿¡ °ËÃâ MycoAlert PLUS mycoplasma detection kit
- RNP µµÀÔµµ °¡´ÉÇÑ: Guide-it¢â Recombinant Cas9 (Electroporation-Ready)
- sgRNA ÇÕ¼º & ½ºÅ©¸®´×: Guide-it¢â Complete sgRNA Screening System)
½ÇÇè µ¥ÀÌÅÍ

±×¸²2. Ÿ»çÁ¦Ç° ´ëºñ ³ôÀº transfection È¿À²°ú ³·Àº ¼¼Æ÷µ¶¼ºÀ» ³ªÅ¸³»´Â TransIT-X2¢ç Dynamic Delivery System. A549 (A)¿Í MDCK (B) ¼¼Æ÷¿¡ luciferase¸¦ encodingÇÏ´Â plasmid¸¦ TransIT-X2¢ç, Lipofectamine¢ç 2000 (Thermo Fisher Scientific), Lipofectamine¢ç 3000 (Thermo Fisher Scientific)À¸·Î °¢°¢ÀÇ ÃÖÀû Á¶°Ç¿¡¼­ 24½Ã°£ µ¿¾È transfection ÇÑ °á°úÀÌ´Ù. Luciferase activity·Î transfection È¿À²À» ÃøÁ¤ÇÏ°í, ¼Õ»óµÈ ¼¼Æ÷ÀÇ ¼¼Æ÷Áú¿¡¼­ ¹æÃâµÇ´Â LDH¸¦ Á¤·®ÇØ ¼¼Æ÷µ¶¼ºÀ» Æò°¡ÇÏ¿´´Ù. ±× °á°ú Lipofectamine¢ç 2000 ¹× Lipofectamine¢ç 3000¿¡ ºñÇØ TransIT-X2¢ç¿¡¼­ ³ôÀº transgene ¹ßÇöÀ²°ú ³·Àº ¼¼Æ÷µ¶¼ºÀ» È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.


±×¸² 3. ´Ù¾çÇÑ cell-line ¹× primary cell¿¡¼­ °íÈ¿À² GFP ¹ßÇö. A549, CHO-K1, Hep G2, MDCK, LNCaP, PC-12, primary human mammary epithelial cells (HMEC), normal human dermal fibroblasts (NHDF) ¼¼Æ÷¿¡ EGFP¸¦ encodingÇÏ´Â plasmid¸¦ TransIT-X2¢ç Dynamic Delivery System·Î transfection ÇÑ °á°úÀÌ´Ù. 96-well plate¿¡¼­ 0.2~0.4 §¡ÀÇ TransIT-X2¢ç·Î 0.1 §¶ÀÇ DNA¸¦ transfection Çß°í (2:1, 3:1, 4:1 / reagent: DNA ratio), 48½Ã°£ µÚ 3¹Ýº¹À¸·Î guava easyCyte¢â 5HT Flow Cytometer¿¡¼­ ºÐ¼®µÇ¾ú´Ù. ±× °á°ú TransIT-X2¢ç Dynamic Delivery SystemÀº ´Ù¾çÇÑ cell-line ¹× primary cell¿¡¼­ °íÈ¿À²·Î GFP¸¦ ¹ßÇö½Ãų ¼ö ÀÖ¾ú´Ù.
* Primary cell type


±×¸² 4. Ÿ»çÁ¦Ç° ´ëºñ RNPÀÇ transfection È¿À²ÀÌ ³ôÀº TransIT-X2¢ç Dynamic Delivery System. HEK293T/17°ú U2OS ¼¼Æ÷¿¡ 2-part gRNA (IDT)¸¦ Ÿ°ÙÇÏ´Â PPIB (cyclophilin B)¿Í Cas9 protein (PNA Bio)À¸·Î Á¦ÀÛÇÑ Ribonucleoprotein (RNP) complexes¸¦ TransIT-X2¢ç Dynamic Delivery System (1 §¡/well) ¶Ç´Â Lipofectamine¢ç CRISPRMAX¢â (1.5 §¡/well and 1 §¡/well of Lipofectamine¢ç Cas9 Plus¢â Reagent, ThermoFisher), Lipofectamine¢ç RNAiMAX (1.5 §¡/well), Lipofectamine¢ç 3000 (1.5 ¥ìl/well and 1 ¥ìl/well of P3000¢â Reagent, ThermoFisher)·Î Á¦Á¶»çÀÇ ÇÁ·ÎÅäÄÝ¿¡ µû¶ó transfection ÇÑ °á°úÀÌ´Ù. 6 nM Cas9 protein (PNA Bio)°ú ´Ù¾çÇÑ ¾çÀÇ gRNA (6 nM, 12 nM)·Î Å×½ºÆ®µÇ¾ú°í, transfection 48½Ã°£ ÈÄ T7E1 mismatch detection assay·Î cleavage efficiency¸¦ ÃøÁ¤ÇÏ¿´´Ù. ±× °á°ú TransIT-X2¢ç Dynamic Delivery SystemÀº Ÿ»ç ´ëºñ ³ôÀº È¿À²·Î RNP¸¦ µµÀÔÇÏ¿© °íÈ¿À²·Î À¯ÀüÀÚ ÆíÁýÀ» ÁøÇàÇÒ ¼ö ÀÖ¾ú´Ù.

Keyword : v6003,v6004,v6000,v6005,v6006,mir6003,mir6004,mir6000,mir6005,mir6006,CRISPR,Cas9

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