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Quantifying the transcriptional output of single alleles in single living mammalian cells.

ABSTRACT: Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization in which genes appear as single alleles. Here we present a robust technique for the quantification of mRNA synthesis from a single allele in real time in single living mammalian cells. The protocol describes how to generate cell clones harboring an MS2-tagged allele and how to detect in vivo transcription from this tagged allele at high spatial and temporal resolution throughout the cell cycle. Quantification of nascent mRNAs produced from the single tagged allele is performed using RNA fluorescence in situ hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling detailed in the protocol include measurements of transcription rates of RNA polymerase II, determination of the number of polymerases recruited to the tagged allele and measurement of the spacing between polymerases. Generation of the cells containing the single tagged alleles should take up to 1 month; RNA FISH or live-cell imaging will require an additional week.

SUBMITTER: Yunger S

PROVIDER: S-EPMC3597184 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Quantifying the transcriptional output of single alleles in single living mammalian cells.

Yunger Sharon S Rosenfeld Liat L Garini Yuval Y Shav-Tal Yaron Y

Nature protocols 20130201 2

Transcription kinetics of actively transcribing genes in vivo have generally been measured using tandem gene arrays. However, tandem arrays do not reflect the endogenous state of genome organization in which genes appear as single alleles. Here we present a robust technique for the quantification of mRNA synthesis from a single allele in real time in single living mammalian cells. The protocol describes how to generate cell clones harboring an MS2-tagged allele and how to detect in vivo transcri ...[more]

PMID: 23424748

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Project description:Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors.

Dataset Information

Quantifying the transcriptional output of single alleles in single living mammalian cells.

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Quantifying the transcriptional output of single alleles in single living mammalian cells.

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