title: Imaging Single mRNA Dynamics in Live Neurons and Brains.
abstract: RNA is a key player in the process of gene expression. Whereas fluorescence in situ hybridization allows single mRNA imaging in fixed cells, the MS2-GFP labeling technique enables the observation of mRNA dynamics in living cells. Recently, two genetically engineered mouse models have been developed for the application of the MS2-GFP system in live animals. First, the Actb-MBS mouse was generated by knocking in 24 repeats of the MS2 stem-loop sequence in the 3' untranslated region of the β-actin gene. Second, the MCP mouse was made to express the NLS-HA-MCP-GFP transgene in all cell types. By crossing Actb-MBS and MCP mice, a double homozygous mouse line, MCP×MBS, was established to visualize endogenous β-actin mRNA labeled with multiple green fluorescent proteins. By imaging hippocampal neurons or brain slices from MCP×MBS mice, the dynamics of mRNA, such as transcription, transport, and localization, can be studied at single mRNA resolution. In this chapter, we explain the basics of MCP×MBS mice and describe methods for utilizing these animals.
authors.affiliation: Seoul National University, Seoul, South Korea.
authors.forename: H C
authors.initials: HC
authors.lastname: Moon
authors.affiliation: Seoul National University, Seoul, South Korea. Electronic address: hyeyoon.park@snu.ac.kr.
authors.forename: H Y
authors.initials: HY
authors.lastname: Park
mesh_terms: D020413:3' Untranslated Regions; D000199:Actins / Q000235:genetics; D000818:Animals; D001921:Brain / Q000166:cytology* / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics; D017909:Levivirus / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008867:Microtomy / Q000379:methods; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D020413:3' Untranslated Regions; D000199:Actins; D036022:Capsid Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins
keywords: Brain slice; MS2-GFP; Neuron; Single-molecule imaging; Transcription; Two-photon microscopy; mRNA; mRNA localization; β-Actin