- ´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» ÃÖ¼ÒÈÇÏ¸é¼ 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¤
ÆÐ³Î E: Transduction (16~24½Ã°£ incubation)ÈÄ ½ºÆÝÁö¸¦ 15 §¢ Æ©ºê·Î ¿Å±â´Â °úÁ¤
Lenti-X Transduction Sponge Àû¿ë cell types
º» Á¦Ç°ÀÇ ÀåÁ¡Àº ÀϹÝÀûÀ¸·Î »ç¿ëµÇ´Â cell line ¹× hard-to-transductionÇÑ primary cell¸¦ Æ÷ÇÔÇÏ¿© ´Ù¾çÇÑ ¼¼Æ÷ À¯Çü¿¡ Àû¿ëÇÒ ¼ö ÀÖ°í µ¿ÀÏÇÑ ÇÁ·ÎÅäÄÝÀ» °£ÆíÇÏ°Ô Àû¿ëÇÒ ¼ö ÀÖ´Ù´Â °ÍÀÌ´Ù.
Species |
Origin/tissue type |
Cell type |
Test ±â°ü |
Human |
B-cell lymphoma |
Raji |
TBUSA |
Human |
Cord blood |
CD34+ HSC (primary) |
TBUSA & ¿ÜºÎ Çù·Â¾÷ü |
Human |
Embryonic kidney |
HEK-293T |
¿ÜºÎ Çù·Â¾÷ü |
Human |
Foreskin |
BJ |
TBUSA |
Human |
Hepatocyte carcinoma |
HepG2 |
TBUSA |
Human |
Monocyte |
THP-1 |
TBUSA & ¿ÜºÎ Çù·Â¾÷ü |
Human |
Monocyte |
U937 |
TBUSA |
Human |
Myeloid leukemia |
K562 |
TBUSA |
Human |
Myoblast |
C2C12 |
TBUSA |
Human |
PBMC |
CD3+ T cell (primary) |
TBUSA and TBJapan |
Human |
PBMC |
LCL |
¿ÜºÎ Çù·Â¾÷ü |
Human |
PBMC |
NK (primary) |
TBUSA |
Human |
Retinal pigment epithelial |
RPE-1 |
TBUSA |
Human |
Sarcoma line |
HT1080 |
TBUSA & ¿ÜºÎ Çù·Â¾÷ü |
Human |
T cell |
Jurkat |
TBUSA |
Human |
T lymphoblast |
CCRF-CEM |
TBUSA |
Mouse |
Breast |
4T1 |
¿ÜºÎ Çù·Â¾÷ü |
Mouse |
Fibroblast |
Wehi-13 |
TBUSA |
Mouse |
Hematopoietic stem |
LSK cell |
¿ÜºÎ Çù·Â¾÷ü |
Mouse |
Mast cell |
11PO-1 |
TBUSA |
Mouse |
Pancreas |
Beta TC-6 |
¿ÜºÎ Çù·Â¾÷ü |
Mouse |
T cell |
EL-4 |
TBUSA |
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¸¦ ÆÐ³Î A¿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¯ÁöÇÏ¸é¼ transduction È¿À²À» ³ôÀÏ ¼ö ÀÖ´Ù.
A. Human primary T cellÀ» MHC tetramer, antiCD3/CD28-coated bead, RetroNectin+CD3, CD3¸¦ °¢°¢ »ç¿ëÇÏ¿© Ȱ¼ºÈ½ÃÄ×°í, 48½Ã°£ µÚ CD69+ÀÇ ¹ßÇöÀ» ÃøÁ¤ÇÏ¿© Ȱ¼ºÈ 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 transductionÇϰí, 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)