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shRNA ¹ßÇö¿ë Retrovirus vectors

pSINsi vector ½Ã¸®Áî

-

Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Takara
3660
pSINsi-hH1 DNA
°ü·ÃÇмú ±¸¸ÅÇϱâ
20 ¥ìg
326,400¿ø 
408,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3660_DS.v0802Da.pdf
Takara
3661
pSINsi-hU6 DNA
°ü·ÃÇмú ±¸¸ÅÇϱâ
20 ¥ìg
326,400¿ø 
408,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3661_DS.v0802Da.pdf
Takara
3662
pSINsi-mU6 DNA
°ü·ÃÇмú ±¸¸ÅÇϱâ
20 ¥ìg
326,400¿ø 
408,000¿ø
°¡°ÝÇÒÀÎ
09.02 ~ 10.31
3662_DS.v0802Da.pdf

Á¦Ç° ¼³¸í
pSINsi º¤ÅÍ ½Ã¸®Áî´Â self-inactivated retro viral vector plasmid¸¦ ±âº» °ñ°ÝÀ¸·Î ÇÏ¿© RNA polymerase III (pol III) °èÀÇ promoter¿¡ ÀÇÇØ hairpinÇü RNA¸¦ ¹ßÇö½ÃÅ°´Â vectorÀÌ´Ù. pol III °è¿­ÀÇ promoter ÇÏ·ùÀÇ cloning site ¿¡ hairpinÇü RNA¸¦ ¹ßÇö½ÃÅ°±â À§ÇÏ¿© ÇÕ¼º ¿Ã¸®°í DNA ¹è¿­À» »ðÀÔÇÏ´Â °ÍÀ¸·Î siRNA ¹ßÇö retroviral vector plasmid¸¦ ¾òÀ» ¼ö ÀÖ´Ù. ¼±Åà ¸¶Ä¿·Î neomycine ³»¼º À¯ÀüÀÚ¸¦ °¡Áö°í ÀÖ´Ù. SiRNA ¹ßÇö retroviral vector·ÎºÎÅÍ Retrovirus Packaging Kit Eco/Ampho (Code 6160, 6161)¸¦ ÀÌ¿ëÇÏ¿© ÀçÁ¶ÇÕ retrovirus¸¦ Á¶Á¦ÇÒ ¼ö ÀÖ´Ù.
¿ø¸®
pSINsi vector ½Ã¸®ÁîÀÇ retroviral vector plasmid´Â 3'LTR U3¿µ¿ªÀÇ promoter È°¼ºÀÌ °á¼ÕµÇ¾î ÀÖ¾î ¿ªÀü»ç ÈÄ ¿°»öü¿¡ »ðÀÔÇÑ ÇÁ·Î¹ÙÀÌ·¯½º »óÅ¿¡¼­´Â 5¡¯LTRÀÇ promoter È°¼ºÀÌ ¼Ò½ÇµÇ¾î ÇÁ·Î¹ÙÀÌ·¯½º À¯·¡ÀÇ mRNA´Â Àü»çµÇÁö ¾Ê°Ô µÇ¹Ç·Î polIII°èÀÇ promoter·ÎºÎÅÍ hairpinÇü RNA¿Í SV40 promoterÀÇ neomycin ³»¼º À¯ÀüÀÚ¸¸ Àü»çµÈ´Ù. (¾Æ·¡ ±×¸² ÂüÁ¶)


hairpinÇü RNA ¹ßÇö º¤ÅÍ
Çì¾îÇÉ Çü RNA¸¦ ¹ßÇö½ÃÅ°±â À§ÇÑ DNA ÇÕ¼º
HairpinÇü RNA¸¦ ¹ßÇö½ÃÅ°±â À§Çؼ­ LigationÀÇ Á¦ÇÑ È¿¼Ò »çÀÌÆ® + Ç¥Àû ¼­¿­(sence) + ·çÇÁ ¼­¿­ + Ç¥Àû ¼­¿­ (antisence) + Å͹̳×ÀÌÅÍ ¼­¿­ + LigationÀÇ Á¦ÇÑ È¿¼Ò »çÀÌÆ®¸¦ Â÷·Ê·Î µðÀÚÀÎÇÑ ÇÕ¼º DNA ¸¦ promoterÀÇ ÇÏ·ù¿¡ »ðÀÔÇÑ´Ù. pBAsi vector cloning site´Â neomycin ³»¼º ¹× puromycin ³»¼ºÀÇ °æ¿ì »ó·ù ÃøÀº BamH I, ÇÏ·ù ÃøÀº Xba I, Sse8387 I, Hind III Áß Çϳª¸¦ »ç¿ëÇÒ ¼ö ÀÖ´Ù. ¾àÁ¦ ³»¼º À¯ÀüÀÚ¸¦ Æ÷ÇÔÇÏÁö ¾Ê´Â pBAsi º¤ÅÍÀÇ °æ¿ì À§ÀÇ »çÀÌÆ®»Ó¸¸ ¾Æ´Ï¶ó ÇÏ·ù Sal I, Acc I, Hinc II, Pst I, Sph I »ç¿ë ÇÒ ¼ö ÀÖ´Ù.
pol III °è promoterÀÇ Àü»ç °³½Ã Á¡¿¡¼­ Ç¥Àû ¹è¿­ÀÌ G ¶Ç´Â A·Î ½ÃÀÛÇÏÁö ¾Ê´Â °æ¿ì Ç¥Àû ¹è¿­ ¾Õ¿¡ G ¶Ç´Â A¸¦ »ðÀÔÇÑ´Ù.
·çÇÁ ¹è¿­ÀÇ ¿¹·Î´Â CTGTGAAGCCACAGATGGG (Boden et al. Âü°í ¹®Çå 1)ÀÌ º¸°íµÇ¾ú°í GTGTGCTGTCC (Miyagishi et al. Âü°í ¹®Çå 2)µµ À¯¿ëÇÏ´Ù´Â °ÍÀÌ È®ÀεǾú´Ù. ¶ÇÇÑ, ÀÌ¿ÜÀÇ hairpin ·çÇÁ·Î Lee et al (Âü°í ¹®Çå 3) Paddison et al (Âü°í ¹®Çå 4), Paul et al. (Âü°í ÀÚ·á 5), Sui et al (Âü°í ¹®Çå 6)µé¿¡ ÀÇÇØ °¢°¢ ´Ù¸¥ ¼­¿­ÀÌ º¸°í µÇ°í ÀÖ´Ù.
Å͹̳×ÀÌÅÍ ¹è¿­Àº TTTTTT ¹è¿­À» °¡Áø´Ù. (T°¡ 4 °³ °è¼ÓµÇ¸é pol III °è ¸ðÅÍ¿¡ ÀÇÇÑ Àü»ç°¡ ¸ØÃá´Ù). siRNA ¼­¿­ »ç¿ë½Ã hairpin ·çÇÁ ¹è¿­ Á¶ÇÕÀº T°¡ 4 °³ ÀÌ»ó Áö¼ÓµÉ °¡´É¼ºÀÌ Àֱ⠶§¹®¿¡ ÇÕ¼º DNA¸¦ µðÀÚÀÎ ÈÄ ¹Ýµå½Ã Top strand¿¡ T°¡ 4 °³ ÀÌ»ó °è¼ÓµÇÁö ¾Ê¾Ò´ÂÁö È®ÀÎÇÏ´Â °ÍÀÌ ÇÊ¿äÇÏ´Ù.

¡¡¡¡¡¡¡¡Àü»ç½ÃÀÛÁ¡*

BamH¡é ¡¡¡¡I

target sequence (sense)

Hairpin loop

target sequence (antisense)

Terminator

Cla I

Top strand

5'-GATCC(G/A)

NNNNNNNNNNNNNNNNNNN

CTGTGAAGCCACAGATGGG

NNNNNNNNNNNNNNNNNNN

(C/T)

TTTTTT

AT-3'

Bottom strand

3'-G(C/T)

NNNNNNNNNNNNNNNNNNN

GACACTTCGGTGTCTACCC

NNNNNNNNNNNNNNNNNNN

(G/A)

AAAAAA

TAGC-5'

º¸Á¸
-20¡É
Vectormap
pSINsi º¤ÅÍ ½Ã¸®Áî´Â 3°¡Áö ´Ù¸¥ Á¾·ùÀÇ ÇÁ·Î¸ðÅÍ ÇÃ¶ó½º¹Ìµå º¤ÅÍ°¡ ÀÖ´Ù.

pSINsi - hH1 DNA : human H1 promoter (Accession S68670) Æ÷ÇÔ.
pSINsi - hU6 DNA : human U6 promoter (Accession X07425) Æ÷ÇÔ.
pSINsi - mU6 DNA : mouse U6 promoter (Accession X06980) Æ÷ÇÔ.



pSINsi º¤ÅÍÀÇ Á¦ÇÑ È¿¼ÒÁöµµ
°¢ º¤ÅÍÀÇ DNA ½ÃÄö½º µ¥ÀÌÅÍ
pSINsi-hH1 DNA sequence data
DNA sequence data
DNA sequence data
¡¼ÁÖÀÇ¡½ ½ÃÄö½º¿¡¼­ 1259 À§Ä¡ (ÇÁ·Î¸ðÅÍ »ó·ù, Hind III¿Í EcoR I »çÀÌ) Cla I »çÀÌÆ®°¡ ÀÖÁö¸¸ ÀÏ¹Ý dam + ´ëÀå±Õ¿¡¼­ Á¶Á¦ÇÑ Çö󽺹̵å´Â dam methylaseÀÇ ¿µÇâÀ» ¹Þ±â ¶§¹®¿¡ 1259 À§Ä¡ ÀÇ Cla I »çÀÌÆ®´Â Àý´ÜµÇÁö ¾Ê½À´Ï´Ù.

Keyword :

siRNA ¹ßÇö ¡¤ ºÐ¼®
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shRNA ¹ßÇö¿ë Retrovirus vectors
pSINsi vector ½Ã¸®Áî
>
Åõ¿©ÇÑ ÇÕ¼º siRNAÀÇ ÃøÁ¤¿¡
Synthetic siRNA Quantitation Kit