Imaging cell-type-specific dynamics of mRNAs in living mouse brain

Chiso Nwokafor; Robert H. Singer; Hyungsik Lim

doi:10.1016/j.ymeth.2018.07.009

Imaging cell-type-specific dynamics of mRNAs in living mouse brain

Chiso Nwokafor, Robert H. Singer, Hyungsik Lim

Cell Biology

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.

Original language	English (US)
Pages (from-to)	100-105
Number of pages	6
Journal	Methods
Volume	157
DOIs	https://doi.org/10.1016/j.ymeth.2018.07.009
State	Published - Mar 15 2019

Keywords

Intravital microscopy
Transcriptional dynamics
Viral transfer
mRNA transport

ASJC Scopus subject areas

Molecular Biology
Biochemistry, Genetics and Molecular Biology(all)

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10.1016/j.ymeth.2018.07.009

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title = "Imaging cell-type-specific dynamics of mRNAs in living mouse brain",

abstract = "We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.",

keywords = "Intravital microscopy, Transcriptional dynamics, Viral transfer, mRNA transport",

author = "Chiso Nwokafor and Singer, {Robert H.} and Hyungsik Lim",

note = "Funding Information: We thank Xiuhua Meng for help with cloning and Dr. Vladislav Verkhusha for advice on far-red fluorescent proteins. C.N. received support from the MBRS program at Hunter College, funded by National Institute of Health ( GM060665 ). This work was supported by funds from the National Institute of Health ( EB013571 to H.L. and R.H.S., NS083085 to R.H.S. and GM121198 to H.L.). Publisher Copyright: {\textcopyright} 2018 Elsevier Inc.",

year = "2019",

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doi = "10.1016/j.ymeth.2018.07.009",

language = "English (US)",

volume = "157",

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AU - Nwokafor, Chiso

AU - Singer, Robert H.

AU - Lim, Hyungsik

N1 - Funding Information: We thank Xiuhua Meng for help with cloning and Dr. Vladislav Verkhusha for advice on far-red fluorescent proteins. C.N. received support from the MBRS program at Hunter College, funded by National Institute of Health ( GM060665 ). This work was supported by funds from the National Institute of Health ( EB013571 to H.L. and R.H.S., NS083085 to R.H.S. and GM121198 to H.L.). Publisher Copyright: © 2018 Elsevier Inc.

PY - 2019/3/15

Y1 - 2019/3/15

N2 - We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.

AB - We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for investigating the cell-type-dependent dynamics of mRNAs underlying the structure and function of the brain.

KW - Intravital microscopy

KW - Transcriptional dynamics

KW - Viral transfer

KW - mRNA transport

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SP - 100

EP - 105

JO - ImmunoMethods

JF - ImmunoMethods

ER -

Imaging cell-type-specific dynamics of mRNAs in living mouse brain

Abstract

Keywords

ASJC Scopus subject areas

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