RRC ID 67391
Author Luo W, Mizuno H, Iwata R, Nakazawa S, Yasuda K, Itohara S, Iwasato T.
Title Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo.
Journal Sci Rep
Abstract Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.
Volume 6
Pages 35747
Published 2016-10-24
DOI 10.1038/srep35747
PII srep35747
PMID 27775045
PMC PMC5075795
MeSH Animals Clustered Regularly Interspaced Short Palindromic Repeats / genetics Electroporation / methods Female Gene Knockdown Techniques / methods Gene Knockout Techniques / methods Gene Transfer Techniques Genetic Vectors / genetics* Hippocampus / metabolism Humans Male Mice Neurons / metabolism Recombination, Genetic / genetics Single-Cell Analysis / methods
IF 3.998
DNA material pK031.TRE-Cre (Supernova) (RDB15043) pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) (RDB15045) pK036.TRE-Flpe-WPRE (Supernova) (RDB15046) pK068.CAG-FRT-stop-FRT-EGFP-ires-tTA-WPRE (Supernova) (RDB15047) pK170.AAV-TRE-Cre-WPRE (Supernova) (RDB15050) pK237.U6-Chimeric-CAG-loxP-stop-loxP-hspCas9 (Supernova) (RDB15051) pK073.TRE-Dre-WPRE (Supernova) (RDB15053) pK224.CAG-Rox-STOP-Rox-GFP-ires-tTA-WPRE (Supernova) (RDB15055) pK039.CAG-loxP-stop-loxP-AmCyan-ires-tTA-WPRE (Supernova) (RDB15056) pK238.U6-gRNACreb1-CAG-loxP-stop-loxP-hspCas9 (Supernova) (RDB15057) pK177.CAG-loxP-stop-loxP-mir30 (GFP RNAi) (Supernova) (RDB15058) pK178.CAG-loxP-stop-loxP-mir30 (GFP RNAi scramble control) (Supernova) (RDB15059) pK229.AAV-EF1alpha-EGFP-WPRE (Supernova) (RDB15061)