Figure 1
From: Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo
IUE-based Supernova enables sparse and bright cell labeling with little background.
(a) Schematics showing the elementary components of IUE-based Supernova: TRE-SSR-WPRE-pA (TRE-SSR) and CAG-RT-stop-RT-XFP-ires-tTA-WPRE-pA (CAG-RT-stop-RT-XFP-tTA). TRE: tetracycline response element; tTA: tetracycline transactivator; SSR: site specific recombinase (e.g. Cre, Flpe); RT: recombination target site (e.g. loxP, FRT); XFP: fluorescent proteins (e.g. GFP, RFP); WPRE: WHP post-trascriptional response element; ires: internal ribosome entry site. (b) Schematics showing how Supernova works: (1) Initially, only in a sparse population among many cells that are transfected with both vectors, the leakage of TRE drives above-threshold but weak SSR expression. (2) This low level of SSR excises the RT-stop-RT cassette in a few copies of CAG-RT-stop-RT-XFP-tTA vector, initiating the transcription of XFP and tTA, albeit weakly. (3) Through binding with TRE, tTA facilitates expression of SSR. (4) Then RT-stop-RT cassette is excised from many copies of CAG-RT-stop-RT-XFP-tTA vector, and expression of XFP and tTA is increased. This positive loop of tTA/TRE enhancement (See Supplementary Fig. 3) leads to extremely high levels of expression of both SSR and XFP, only in a small population of transfected cells. (c–g) Supernova labeling is sparse and bright enough to visualize the detailed morphology of single neurons including dendritic spines (f) and axonal branches and boutons (g). An Flpe-based Supernova GFP (Flpe-SnGFP) vector set (TRE-Flpe and CAG-FRT-stop-FRT-GFP-tTA) was introduced into layer 2/3 (L2/3) cortical neurons by in utero electroporation (IUE). The CAG-RFP vector was co-electroporated to mark the transfected neurons. Higher-power images of the rectangles in c were shown in (d). The square in (d) were further magnified in (e). (f,g) Show higher-magnification images of the rectangles in (e). (h) Two-photon in vivo imaging of L4 cortical neurons labeled by Flpe-based Supernova RFP (Flpe-SnRFP) in P5 mouse. The traces of imaged cortical neurons were shown in right panel. Black lines indicate the dendrites of labeled neurons. The axons of these neurons are represented by red and blue lines, separately. Scale bars, 250 μm (c); 100 μm (d); 50 μm (e,h); 4 μm (f); 10 μm (g).
