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Home > ÀüÁ¦Ç°º¸±â > Gene transfer (virus) > Lentivirus > Lenti-X¢â Transduction Sponge
½Å°³³ä lentivirus transduction

Lenti-X¢â Transduction Sponge

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
631478
Lenti-X¢â Transduction Sponge
°ü·ÃÇмú ±¸¸ÅÇϱ⠶óÀ̼±½º 
24 ȸ
995,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x347949

  • ´Ù°ø¼º ½ºÆÝÁö·Î ºü¸£°í È¿°úÀûÀ¸·Î lentivirus transduction È¿À² Áõ´ë
  • ½ºÆÝÁö Çϳª·Î 24 well plate Å©±âÀÇ 1 well¿¡¼­ 105~107 cell¿¡ transduction °¡´É
  • Spinoculation°ú polybreneÀ» »ç¿ëÇÏÁö ¾Ê¾Æ ¼¼Æ÷ ¼Õ»óÀÌ ÀûÀ½
  • TransductionÀÌ ¾î·Á¿î primary cell ¹× ´Ù¾çÇÑ cell-line¿¡ Àû¿ë °¡´É
  • ÀÛÀº reaction volumeÀ¸·Î ¼Ò·®ÀÇ ¹ÙÀÌ·¯½º·Îµµ ½ÇÇè °¡´É
Á¦Ç°¼³¸í
Lenti-X¢â Transduction Sponge´Â ´Ù°ø¼º alginate (¾Ë±ä»ê)·Î ±¸¼ºµÈ ½ºÆÝÁö·Î °£ÆíÇÏ°Ô lentivirus transductionÀ» ÃËÁøÇÑ´Ù. ½ºÆÝÁöÀÇ ´Ù°ø¼º microfluidics ±¸Á¶´Â ¼¼Æ÷¿Í lentivirus°£ ¹°¸®Àû °Å¸®¸¦ Á¼Çô spinoculationÀ̳ª chemical enhancer (e.g. Polybrene) ¾øÀ̵µ ¼¼Æ÷ µ¶¼ºÀ» ÃÖ¼ÒÈ­Çϸ鼭 ºü¸¥ ½Ã°£ ¾È¿¡ È¿°úÀûÀ¸·Î transductionÀ» ÁøÇàÇÑ´Ù. 24 well plate Å©±âÀÇ ½ºÆÝÁö´ç Çѹø¿¡ 105~107 cellÀ» transductionÇÒ ¼ö ÀÖÀ¸¸ç º°µµÀÇ microfluidics ±â±â ¾øÀÌ ÇüÁúµµÀÔÀÌ ¾î·Á¿î cell-line (ºÎÂøÇü, ºÎÀ¯Çü ¼¼Æ÷ ¸ðµÎ °¡´É) ¹× primary cell µî ´Ù¾çÇÑ ¼¼Æ÷¿¡ transductionÀ» ÁøÇàÇÒ ¼ö ÀÖ´Ù.
cell handlingÀÌ ÂªÀº °£ÆíÇÑ workflow, Mix-Drop-Spin

±×¸² 1. Lenti-X¢â Transduction Sponge workflow
1. Ÿ°Ù ¼¼Æ÷¿Í lentivirus¸¦ È¥ÇÕ (ÃÑ ºÎÇÇ ÃÖ´ë 150 §¡).
2. ½ºÆÝÁö¸¦ 24well plate·Î well´ç 1°³¾¿ ¿Å±â°í 1¹øÀÇ mixture¸¦ ½ºÆÝÁö À§·Î dropwise ¹æ½ÄÀ¸·Î ÷°¡ ¹× Èí¼ö.
3. 37 ¡ÉÀÇ CO2 ÀÎÅ¥º£ÀÌÅÍ¿¡¼­ 1½Ã°£µ¿¾È incubation ÈÄ well¿¡ ¹èÁö¸¦ Ãß°¡·Î 1 §¢ ÷°¡, ÀÌÈÄ ÀÎÅ¥º£ÀÌÅÍ¿¡¼­ 16~24½Ã°£ ¹è¾ç.
4. ´ÙÀ½³¯ ¹èÁö¿Í ½ºÆÝÁö¸¦ ¸ðµÎ 15 §¢ Æ©ºê·Î ¿Å±â°í Release Buffer 1 §¢¸¦ ÷°¡ ÈÄ ¿ø½ÉºÐ¸®ÇÏ¿© ½ºÆÝÁö¸¦ ¿ëÇØ, ¿ëÇØ ÈÄ ¹æÃâµÈ ¼¼Æ÷¸¦ PBS·Î 2ȸ wash.
5. TransductionµÈ ¼¼Æ÷¸¦ »õ culture vessel·Î À̵¿ ÈÄ ¹è¾ç.


±×¸² 2. Lenti-X¢â Transduction Sponge¿Ü°ü ¹× »ç¿ë¹ý
A: Á¦Ç° »çÁø (°³º° Æ÷ÀåµÈ ½ºÆÝÁö 24°³)
B: ´Ù°ø¼º ½ºÆÝÁöÀÇ È®´ë»çÁø (150¹è), 20~300 §­ÀÇ microfluidics ±¸Á¶
C: ÇɼÂÀ¸·Î ½ºÆÝÁö¸¦ 24 well plate·Î ¿Å±â´Â °úÁ¤
D: ½ºÆÝÁö¿¡ ¼¼Æ÷¿Í ¹ÙÀÌ·¯½º mixture¸¦ ÷°¡ÇÑ µÚ 1½Ã°£ µ¿¾È incubationÇÏ´Â °úÁ¤
ÆгΠE: Transduction (16~24½Ã°£ incubation)ÈÄ ½ºÆÝÁö¸¦ 15 §¢ Æ©ºê·Î ¿Å±â´Â °úÁ¤
½ÇÇè µ¥ÀÌÅÍ

±×¸² 3. Lenti-X¢â Transduction SpongeÀº ´Ù¾çÇÑ cell¿¡¼­ spinoculation (with polybrene)°ú µ¿µîÇϰųª ´õ È¿°úÀûÀÎ transductionÀÌ °¡´É.
A: 1 X 106 Jurkat ¼¼Æ÷¿¡ spinoculation°ú Lenti-X¢â Transduction Sponge ¹æ¹ýÀ¸·Î ZsGreen1À» ¹ßÇöÇÏ´Â lentivirus¸¦ MOI 0, 1, 5, 10·Î transductionÇÑ °á°úÀÌ´Ù. SpinoculationÀº cell plate¸¦ Polybrene 8 §¶/§¢°ú ÇÔ²² 32¡É¿¡¼­ 1,400 x g·Î 90ºÐ°£ ¿ø½ÉºÐ¸®ÇÏ¿´°í, Lenti-X¢â Transduction Sponge´Â Á¦Ç°ÀÇ »ç¿ëÀÚ¸Þ´º¾ó¿¡ µû¶ó ÁøÇàÇÏ¿´´Ù. µÎ ¹æ¹ý ¸ðµÎ transduction 48½Ã°£ µÚ ZsGreen1ÀÇ ¹ßÇöÀ²À» FCM (Flow Cytometry)À¸·Î ºÐ¼®ÇÏ¿© Á¤¸®ÇÏ¿´´Ù. ±× °á°ú Lenti-X¢â Transduction Sponge°¡ spinoculation ¹æ¹ýº¸´Ù °£ÆíÇÏ°í ´õ¿í È¿°úÀûÀ̾ú´Ù.
B: ZsGreen1 ¹ßÇö lentivirus¸¦ ÆгΠA¿Í µ¿ÀÏÇÑ ¹æ¹ýÀ¸·Î ´Ù¾çÇÑ cell line¿¡¼­ transductionÇÑ °á°úÀÌ´Ù. ´Ù¾çÇÑ ¼¼Æ÷¿¡¼­ Lenti-X¢â Transduction Sponge´Â spinoculation ¹æ¹ý°ú µ¿µîÇϰųª ´õ È¿°úÀûÀ̾ú´Ù.


±×¸² 4. Lenti-X¢â Transduction Sponge´Â primary T cellÀÇ viability¸¦ À¯ÁöÇϸ鼭 transduction È¿À²À» ³ôÀÏ ¼ö ÀÖ´Ù.
A. Human primary T cellÀ» MHC tetramer, antiCD3/CD28-coated bead, RetroNectin+CD3, CD3¸¦ °¢°¢ »ç¿ëÇÏ¿© È°¼ºÈ­½ÃÄ×°í, 48½Ã°£ µÚ CD69+ÀÇ ¹ßÇöÀ» ÃøÁ¤ÇÏ¿© È°¼ºÈ­ Á¤µµ¸¦ ÃøÁ¤ÇÏ¿´´Ù. ÀÌÈÄ 1 x 106 °³ÀÇ È°¼ºÈ­µÈ ¼¼Æ÷¿¡ Lenti-X¢â Transduction Sponge¸¦ »ç¿ëÇØ 24½Ã°£ µ¿¾È ZsGreen1À» ¹ßÇöÇÏ´Â lentivirus·Î transductionÇÏ°í, transduction 48½Ã°£ ÈÄ FACS·Î ZsGreen1ÀÇ ¹ßÇöÀ²À» ÃøÁ¤ÇÏ¿© %·Î Á¤¸®ÇÑ °á°úÀÌ´Ù.
B. Transduction 48½Ã°£ ÈÄ 7-AAD ¿°»ö°ú FACS¸¦ »ç¿ëÇØ viability¸¦ ÃøÁ¤ÇÑ °á°ú, ³ôÀº viability¸¦ È®ÀÎÇÒ ¼ö ÀÖ¾ú´Ù.
º¸Á¸: 4¡É (Lenti-X¢â Transduction Sponges´Â °ÇÁ¶Á¦¿Í ÇÔ²² ÆÄ¿ìÄ¡ º¸°ü)
³»¿ë

Lenti-X¢â Transduction Sponges

24

Release Buffer

30 §¢

Forceps

1

°ü·ÃÁ¦Ç°
- CGT ¿¬±¸¿¡ ÁÖ·Î »ç¿ëµÇ´Â cellµé¿¡ Àû¿ë °¡´ÉÇÑ transduction enhancer: RetroNectin¢ç (Code T100A)
- Lentivirus ³óÃà, ÀϹݿø½ÉºÐ¸®±â·Î 1½Ã°£¸¸¿¡ 100¹è: Lenti-X¢â Concentrator (Code 631231 ¿Ü)
- ÀçÇö¼º ÀÖ´Â ½ÇÇèÀ» À§Çؼ­´Â titrationÀÌ ÇʼöÀÔ´Ï´Ù.
    qPCR¹ý: Lenti-X¢â qRT-PCR Titration Kit (Code 631235)
    ELISA¹ý: Lenti-X¢â p24 Rapid Titer Kit (Single Wash) (Code 631476)
    Lateral flow¹ý Á¤·®, 10ºÐ ¿Ï¼º: Lenti-X¢â GoStix¢â Plus (Code 631280)




Keyword :

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