Fig. 7: Astrocyte specificity of the 4x6T cassette with inducible and alternative recombinases.

a Tamoxifen- (ERCreER) and light- (iCreV) inducible forms of Cre show high levels of neuronal background without induction when co-injected with a flexV5 reporter with no 4x6T cassette (PHP.eB::CAG-flex-smV5) but much less background when co-injected with a flexV5-4x6T reporter (PHP.eB::CAG-flex-smV5-4x6T). Scale bars: 100 μm. Astrocyte specificity with a flexV5-4x6T reporter is high with both inducible forms of Cre, but higher in ERCreER (two-tailed unpaired t test, **P = 0.0055, t = 4.233, df=6; n = 4 mice with tamoxifen or light; two mice with no tamoxifen or no light). Light induction of iCreV shows expression across a broad area of cortex ipsilateral to light placement and limited contralateral expression. Light placement: yellow arrow. Scale bar: 500 μm. All: 2–5-month-old mice, euthanized 2 weeks after Cre induction, 4–4.5 weeks after retroorbital virus injection. Titers: ERCreER-4x6T, iCreV-4x6T: 5 × 10¹¹ vg/mouse + CAG-flex-smV5-4x6T: 5 × 10¹¹ vg/mouse. b PHP.eB::GfaABC1D-Dre-4x6T and Dre-dependent reporter PHP.eB::CAG-dDIO-smMyc-4x6T can be used orthogonally with Cre/flex viral systems and show similar levels of astrocytic specificity (scale bar: 50 μm); yellow box shows the region of higher magnification on the right (scale bar: 10 μm); n = 4 mice per recombinase, 2–3-month-old mice, euthanized 2 weeks after retroorbital virus delivery. Titers: Dre-4x6T: 2 × 10¹¹ vg/mouse; Cre-4x6T, dDIO-smMyc-4x6T, flex-smV5-4x6T: 1 × 10¹¹ vg/mouse. Source data are provided as a Source Data file. All data are presented as mean ± SEM. c Schematic diagrams of empty vectors with multiple cloning sequences (MCS) for insertion of other cargo: GfaABC1D-MCS--4x6T for non-recombinase-dependent expression; CAG-flex-MCS--4x6T for Cre-dependent expression; and CAG-dDIO-MCS--4x6T for Dre-dependent expression. Note the presence of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); while we omitted this element in the case of recombinase vectors, where high levels of transgene expression were neither wanted nor needed, we have included it in these more general vectors. More detail on transgene components and the full sequences can be found on Addgene.