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A transgenic mouse for in vivo detection of endogenous labeled mRNA.

ABSTRACT: Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential ?-actin gene. As ?-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the ?-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single ?-actin mRNA molecules in various mouse tissues.

SUBMITTER: Lionnet T

PROVIDER: S-EPMC3076588 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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A transgenic mouse for in vivo detection of endogenous labeled mRNA.

Lionnet Timothée T Czaplinski Kevin K Darzacq Xavier X Shav-Tal Yaron Y Wells Amber L AL Chao Jeffrey A JA Park Hye Yoon HY de Turris Valeria V Lopez-Jones Melissa M Singer Robert H RH

Nature methods 20110116 2

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely addre ...[more]

PMID: 21240280

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Project description:IntroductionThe accumulation of amyloid-? is a pathological hallmark of Alzheimer's disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-? antibody (JRF/A?N/25) to non-invasively assess amyloid-? burden in aged transgenic mice (APPPS1-21) with ?PET imaging.MethodsWe investigated the antibody JRF/A?N/25 that binds to full-length A?. JRF/A?N/25 was radiolabeled with a [(89)Zr]-desferal chelate and intravenously injected into 12-13 month aged APPPS1-21 mice and their wild-type (WT) controls. Mice underwent in vivo ?PET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [(89)Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/A?N/25 and finally we assessed the possible confounding effects of blood retention.ResultsVoxel-wise analysis of ?PET data demonstrated significant [(89)Zr]-Df-Bz-JRF/A?N/25 retention in APPPS1-21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [(89)Zr]-Df-Bz-JRF/A?N/25 was found at 4 and 7 days pi in APPPS1-21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/A?N/25 only partially blocked [(89)Zr]-Df-Bz-JRF/A?N/25 uptake indicative of a high contribution of non-specific binding.ConclusionAmyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [(89)Zr]-Df-Bz-JRF/A?N/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

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A transgenic mouse for in vivo detection of endogenous labeled mRNA.

Publications

A transgenic mouse for in vivo detection of endogenous labeled mRNA.

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