Photo-activatable Cre recombinase regulates gene expression in vivo

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Abstract

Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods.

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A mesoscale connectome of the mouse brain.

Seung Wook Oh, Julie Harris, Lydia Ng … (2014)

Comprehensive knowledge of the brain's wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.

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Optical Control of Mammalian Endogenous Transcription and Epigenetic States

Silvana Konermann, Mark Brigham, Alexandro E Trevino … (2013)

The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions 1-11 . However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain 12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility 3,4,6,7,15 . LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

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Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.

Hongtao Liu, Xuhong Yu, Kunwu Li … (2008)

Cryptochromes (CRY) are photolyase-like blue-light receptors that mediate light responses in plants and animals. How plant cryptochromes act in response to blue light is not well understood. We report here the identification and characterization of the Arabidopsis CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 binds to G box (CACGTG) in vitro with a higher affinity than its interaction with other E-box elements (CANNTG). However, CIB1 stimulates FT messenger RNA expression, and it interacts with chromatin DNA of the FT gene that possesses various E-box elements except G box. We propose that the blue light-dependent interaction of cryptochrome(s) with CIB1 and CIB1-related proteins represents an early photoreceptor signaling mechanism in plants.

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Author and article information

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 09 September 2015

Publication date Collection: 2015

Volume: 5

Electronic Location Identifier: 13627

Affiliations

[1 ]Department of Neurology and the Hope Center for Neurological Disorders , St. Louis, MO

[2 ]Department of Anesthesiology and the Washington University Pain Center , St. Louis, MO

[3 ]Department of Anatomy and Neurobiology , St. Louis, MO

[4 ]Division of Biological and Biomedical Sciences, Washington University School of Medicine , St. Louis, MO

[5 ]Department of Pharmacology, University of Colorado School of Medicine , Aurora, CO

[6 ]Center for Alzheimer’s and Neurodegenerative Diseases, University of Texas Southwestern Medical Center , Dallas, TX.

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[a ] marc.diamond@ 123456utsouthwestern.edu

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Publisher Item ID: srep13627

DOI: 10.1038/srep13627

PMC ID: 4563371

PubMed ID: 26350769

SO-VID: b8a83ae5-ca8d-4425-8758-8562b49a5dbc

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This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Photo-activatable Cre recombinase regulates gene expression in vivo

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CRISPR/Cas9 editing in human blood

Most cited references 14

A mesoscale connectome of the mouse brain.

Optical Control of Mammalian Endogenous Transcription and Epigenetic States

Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis.

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