Extended Data Fig. 5: In vivo morphological characterization of the PTEN sensor.
From: Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
a Representative images of G-PTEN fluorescence intensity with or without spCas9 and PTEN gRNA. G-PTEN was modified from rat sequence which differs in one nucleotide to mouse PTEN which matches the gRNA template. Scale bar; 20 μm. b Quantification of the fluorescent intensity of the PTEN biosensor co-expressing PTEN gRNA with the mean of G-PTEN alone, for the rat-based version (0.473 ± 0.01, n = 380) and mouse-based sensor (0.003 ± 0, n = 400). (c) Schematic of experimental design for IUE injections, and in vivo 2pFLIM in adult mice. d Representative 2p in vivo images of CyRFP labeled L2/3 cells, co-expressing spCas9 and scrambled control gRNA (Scr), or a PTEN targeted gRNA, and with co-expression of mEGFP-PTEN-sREACh WT or R14G. Scale bar; 30 μm. e Quantification of neuron soma area in μm2 indicated by CyRFP, for neurons co-expressing scrambled guide RNA (696.6 ± 14.72, n = 62), PTEN gRNA (1646 ± 32.77, n = 93), PTEN gRNA+mEGFP-PTEN-sREACh WT (791.5 ± 14.5, n = 78) and PTEN gRNA + mEGFP-PTEN-sREACh R14G (1634 ± 28.87, n = 74). ns p = 0.0787 and p = 0.1554 for Scrambled gRNA compared to PTEN gRNA + WT and for PTEN gRNA compared to PTEN gRNA + R14G respectively. 4-6 mice for each group. f Representative in vivo images of fluorescently labeled apical dendrites expressing CyRFP, and co-expressing spCas9 and scrambled guide RNA or PTEN targeted gRNA, and co-expressing either mEGFP-PTEN-sREACh WT or R14G. Scale bar; 10 μm. 4-6 mice for each group. g Quantification of spine density per 10 μm for dendrites expressing CyRFP and scrambled guide RNA (5.087 ± 0.161, n = 43), PTEN gRNA (11.72 ± 0.53, n = 26), PTEN gRNA + mEGFP-PTEN-sREACh WT (5.18 ± 0.18, n = 40), and PTEN gRNA + mEGFP-PTEN-sREACh R14G (11.77 ± 0.29, n = 49). ns p = 0.9956 and p = 0.9995 for Scrambled gRNA compared to PTEN gRNA + WT and for PTEN gRNA compared to PTEN gRNA + R14G respectively. 4-6 mice for each group. ‘n’ denotes number of cells, Error bars represent s.e.m. statistical differences for (e, g) were measured using one-way ANOVA followed by post-hoc Tukey’s multiple comparison test. Statistical difference for (b) was measured using unpaired two-tailed student t-test. Representative images represent experiments repeated independently at least 3 times. Significant differences in (b, e, g) produced p < 0.0001, unless otherwise stated in the legend above. **** p < 0.0001.
