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MS2 vector system: Tracing mRNA molecules in living cells

MS2 vector system: Tracing mRNA molecules in living cells. 생명과학부 발생유전학 실험실 김병혁. MS2 system. Invented by Robert H. Singer (2003) Articles 1) Localization of ASH1 mRNA Particles in Living Yeast. Molecular Cell (1998)

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MS2 vector system: Tracing mRNA molecules in living cells

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  1. MS2 vector system:Tracing mRNA molecules in living cells 생명과학부 발생유전학 실험실 김병혁

  2. MS2 system • Invented by Robert H. Singer (2003) • Articles 1) Localization of ASH1 mRNA Particles in Living Yeast. Molecular Cell (1998) 2) Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Current Biology (2003) • A method for visualizing the location and movement of a specific RNA of interest in a living mammalian cell • Transient transfection of two vectors

  3. Two vectors A vector encoding a fusion protein (a fluorescent and an RNA-binding domain) <MS2-GFP protein> <mRNA reporter> promoter MS2 GFP NLS promoter mRNA seq. Repeats of MS2 binding sites A vector encoding the RNA (including a protein binding site)

  4. pMS2-GFP • Eukaryotic RNA polymerase II promoter • Bacteriophage MS2 coat protein sequence • GFP sequence (cf. BFP, YFP, CFP) • Nuclear localization signal (NLS) from SV40 (cf. nuclear export signal, NES) promoter MS2 GFP NLS pMS2-GFP Ampr Ori.

  5. pSL-MS2-6 • pSL1180 backbone (Pharmacia) • Bacteriophage MS2 binding site (repeated x6, x12, x24) • MCS for a specific mRNA sequence promoter mRNA seq. Repeats of MS2 binding sites MCS Ori. pSL-MS2-6 Ampr

  6. GFP GFP Application (1) Transient transfection promoter MS2 GFP NLS pMS2-GFP Transient transfection promoter MS2 GFP NLS pMS2-GFP + promoter mRNA seq. (X6) Repeats of MS2 binding sites pSL-MS2-6 (X24)

  7. GFP Application (2) Transient transfection promoter MS2 GFP NLS pMS2-GFP + + promoter mRNA seq. Repeats of MS2 binding sites mRNA binding proteins pSL-MS2-6 (X6)

  8. Advantage • Well-suited for determining where RNA goes in living cells (cf. FISH) • Applicable to the investigation of any RNA-protein complex involved in RNA processing, nuclear export, or intranuclear targeting • Possible artificiality from a transient transfection Disadvantage

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