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Lentivirus를 이용한 sgRNA, Cas9 gene delivery

Lenti-X™ CRISPR/Cas9 System

제조사	제품코드	제품명	용량	가격 (부가세별도)	비고
Clontech	632629	Lenti-X™ CRISPR/Cas9 System	Each (Package)	1,768,800원 ~~2,211,000원~~	11.01 ~ 12.27
Clontech	632630	pLVX-hyg-sgRNA1 Vector System	10 Rxns (Package)	473,600원 ~~592,000원~~	11.01 ~ 12.27
Clontech	632633	Lenti-X™ Tet-On® 3G CRISPR/Cas9 System	1 System (Package)	2,849,600원 ~~3,562,000원~~	11.01 ~ 12.27

Product index

Overivew
유전자 편집 전 단계
편집 유전자 도입
유전자 편집 후 확인
CRE recombinase
- CRE Recombinase Gesicl..
- AAV Helper Free System..

Cas9 gene과 sgRNA를 Lentivirus를 이용하여 세포 내 도입
Transfection이 어려운 mammalian 세포 (proliferating and non-proliferating cells)에서의 CRISPR/Cas9 Genome editing
Lenti-X™ Packaging Single shots을 이용하여 간단하게 고역가의 Lentivirus 제작
Lenti-X™ Tet-On 3G CRISPR/Cas9 system은 Cas9 발현을 효율적으로 조절이 가능하며, 세포 독성 문제나 Cas9의 지속적인 발현으로 인한 off-target effect 감소에 효과적

제품설명

Lenti-X™ CRISPR/Cas9 System은 lentiviral-mediated CRISPR/Cas9 genome editing을 위한 모든 구성품이 포함된 complete system이다.
본 제품은 고역가의 렌티바이러스를 생산하는 Lenti-X™ Packaging Single Shots과 Cas9 단백질, sgRNA를 발현하는 두 개의 플라스미드로 구성되어있다. 목적 유전자를 타겟하는 sgRNA를 발현하는 pLVX-hgy-sgRNA1 plasmid는 이미 선형화 되어있어 아주 쉽게 sgRNA 서열을 삽입, 라이게이션을 진행할 수 있다. Lentivirus를 이용하여 sgRNA와 Cas9 gene을 삽입하면 일반적으로 Transfection이 어렵다고 알려진 세포주에서의 CRISPR/Cas9 genome editing이 가능하다 (그림 1).

Lenti-X™ Tet-On 3G CRISPR/Cas9 System은 doxycycline을 이용하여 Cas9 발현을 유도할 수 있는 Tet-on 3G inducible expression 제품으로써, doxycycline 처리 시 Cas9 단백질을 발현한다. Cas9 발현을 엄격하게 조절할 수 있기 때문에 영구적인 Cas9 단백질 발현으로 인한 off-target effect나 세포 독성을 줄일 수 있다 (그림 2, 그림 3).

그림 1. Either Jurkat or HT1080 cells were first transduced with LVX-hyg-CD81-sgRNA and then selected for stable integration using hygromycin. Stable clones were then transduced with LVX-puro-Cas9. Stable clones were selected for using puromycin and then screened for CD81 knockout efficiency using FACS. Positive and negative controls of parental cells without Cas9 transduction were done either with or without anti-CD81 antibody.

그림 2. Inducible knockout of CD81 in HEK293 cells. Target cells were first transduced with Tet-On 3G lentivirus and selected with G418. Cas9 induction was then assayed by Western blot using polyclonal anti-Cas9 antibody (Code 632607), diluted 1:1,000, and ECL reagent. Panel A. Western blot results for six independent Tet-On 3G-Cas9-positive cell populations cultured in the absence (-) or presence (+) of 0.5 μg/ml doxycycline, assayed with anti-Cas9 antibody. Panel B. FACS results for three independent Tet-On 3G-Cas9-positive 293T cell populations transduced with CD81-sgRNA lentivirus and cultured in the absence (-Dox) or presence (+Dox) of 0.5 μg/ml doxycycline.

그림 3. Inducible editing of AAVS1in HEK293 cells. Induction of Cas9 expression in six stable Tet-On 3G-Cas9-positive HEK293 cell lines was assayed by qRT-PCR. The Guide-it Mutation Detection Kit (Code 631443) was then used to screen for editing of AAVS1. Panel A. qRT-PCR results for Cas9 expression in uninduced (-Dox) and induced (+Dox) cells for each clone. Panel B. Results of Guide-it resolvase assay for detection of AAVS1 gene editing in uninduced (?) and induced (+) clones.

Applications

Lentivirus-based delivery of a user-defined sgRNA and Cas9 for mammalian genome editing using CRISPR/Cas9 technology

구성품 (자세한 내용은 CoA를 참조하세요)

Lenti-X CRISPR/Cas9 System (Code 632629)

pLVX-hyg-sgRNA1 Vector (Linear) (Vector map / Full sequence (txt.))
pLVX-puro-Cas9 Vector (Vector map / Full sequence (txt.))
Stellar Competent Cells
Guide-it Ligation Components v2
Lenti-X Packaging Single Shots

pLVX-hyg-sgRNA1 Vector System (Code 632630)

pLVX-hyg-sgRNA1 Vector (Vector map / Full sequence (txt.))
Guide-it Ligation Components v2
Stellar Competent Cells

Lenti-X™ Tet-On 3G CRISPR/Cas9 System (Code 632633)

pLVX-hyg-sgRNA1 Vector (Linear) (Vector map / Full sequence (txt.))
pLVX-TRE3G-Cas9-puro Vector Set
- pLVX-TRE3G-Cas9-puro Vector (Vector map / Full sequence (txt.))
- pLVX-TRE3G-Luc Control Vector (Vector map / Full sequence (txt.))
pLVX-EF1a-Tet3G Vector (Vector map / Full sequence (txt.))
Stellar Competent Cells
Guide-it Ligation Components v2
Lenti-X Packaging Single Shots

Keyword : Lenti,Lentivirus,Lentiviral,Tet,Tetracycline,inducible,inducible system,도입효율,CRISPR,CRISPR/Cas9,Cas9,sgRNA,크리스퍼,gene editing,유전자편집

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