Short hairpin RNA (shRNA) – I 편
shRNA 란?
RNAi 현상을 유도하는 loop structure 의 short hairpin RNA로 target mRNA 에 상보적 결합을 하여 mRNA degradation 유도
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Drosha
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Nucleus에서 pri-miRNAs와 shRNAs processing하는 RNase III enzyme
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Dicer
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Ribonuclease (RNase) III enzyme은 dsRNA를 20–25 bp siRNAs로 cleavage하고 3’ 말단에 2nt overhang을 남김
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RISC
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minimal RNA-induced silencing complex (RISC) 는 dsRNA를 인식하는 효소 중합체
① dsRNA를 ssRNA로 풀어주는 Helicase activity기능
② RISC가 Anti-sense를 취해 target mRNA에 가져감
③ nuclease activity 기능으로 target mRNA를 degradation 시키거나 cleavage 시킴
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(1) microRNA-adapted shRNAs
Off-target effect 가능성을 최소화하면서 endogenous RNAi pathway를 통한
효과적인 RNAi를 위해 microRNA scaffold-specific attributes 사용
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• SMARTvector Lentiviral shRNA
1) Human, mouse, rat gene을 target으로 하는 microRNA 기반 shRNA
2) 7개의 promoter 중에서 선택하여 특정 세포에 맞게 SMARTvector shRNA 선택 가능
• SMARTvector Inducible Lentiviral shRNA
1) SMARTvector lentiviral shRNA는 Tet-inducible technology 결합
2) mCMV, PGK, hEF1a, mEF1a 의 inducible promotor 중 선택 가능
• GIPZ Lentiviral shRNA
1) microRNA가 적용된 shRNA 디자인으로 효율적인 gene silencing 가능
2) Human과 mouse lentiviral vector 또는 high-titer lentiviral particle로 제공
• TRIPZ Inducible Lentiviral shRNA
1) SMARTvector lentiviral shRNA는 Tet-inducible technology 결합
2) human을 위한 lentiviral vector constructs 이용 가능
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(2) Simple hairpin shRNAs
21개의 base pair sense, antisense stem의 간단한 hairpin과
6개의 base pair loop를 사용하는classic hairpin, rules-based shRNA design 제공
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• TRC Lentiviral shRNA
Human과 mouse에 적용되는 RNAi Consortium의 Lentiviral shRNA collection
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SMARTvector shRNA
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GIPZ
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TRIPZ
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TRC
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Species
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Human, Mouse, Rat
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Human, Mouse
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Human
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Human, Mouse
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| Promoter | Choice of 7 constitutive* and 4 inducible promoters |
Human CMV Pol II
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TRE-min-CMV
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U6 Pol III
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Vector backbone
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Lentiviral
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Lentiviral
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Lentiviral
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Lentiviral
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Stem-loop format
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MicroRNA-adapted
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MicroRNA-adapted
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MicroRNA-adapted
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Simple
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Fluorescent reporter gene
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GFP/RFP
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GFP
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RFP
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Guaranteed silencing
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In vivo RNAi
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Create stable cell lines
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Recommended for primary
and non-dividing cells
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Inducible expression
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Whole genome library
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Human, Mouse, Rat
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Human, Mouse
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Human
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Human
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Formats
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Bacterial glycerol stock,
High-titer lentiviral particles,
Gene families and pathways
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Bacterial glycerol stock,
Arrayed library format,
Gene families and pathways,
High-titer lentiviral particles, shRNA Starter Kit
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Bacterial glycerol stock,
Arrayed library format,
Gene families and pathways, shRNA Starter Kit
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Bacterial glycerol stock,
Arrayed library format,
Gene families and pathways
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