Fig. 3: Elimination of recombination in FRET sensors and in vivo ratiometric imaging of AAV-delivered FLIIP and cdFLIIP. | Communications Biology

Fig. 3: Elimination of recombination in FRET sensors and in vivo ratiometric imaging of AAV-delivered FLIIP and cdFLIIP.

From: Abundance-biased codon diversification prevents recombination in AAV production and ensures robust in vivo expression of functional FRET sensors

Fig. 3

a Gel electrophoresis of the PCR-amplified viral vector DNAs, showing highly intense recombination bands for FLIIP and ATeam and the absence of recombination for the corresponding codon-diversified versions (cdFLIIP and cdATeam), including a “double-acceptor” version of ATeam (cdATeamDA). b Schematic representation of the viral vector production outcome, showing that upon codon diversification only the full sensor sequence is present. c Ratiometric response of transfected HEK cells expressing FLIIP (red) or cdFLIIP (black) to bath glucose administration. Vertical bars indicate switching of the glucose concentration from 2 to 0 and from 0 to 25 mM. Since transfection is not affected by recombination, the two sensors behave identically. Curves are shown as mean ± SEM (FLIIP: N = 28 cells, n = 3 experiments; cdFLIIP: N = 22, n = 3). d Color-coded ratiometric images for cortical neurons expressing FLIIP or cdFLIIP, under 870 or 925 nm two-photon excitation. The schemes on the right represent the expected excitation and emission processes for the recombining FLIIP vectors. The fact that 925 nm light significantly excites YFPs directly allows better visualization of the cells containing predominantly YFPs. The red arrow highlights the presence of darker circular areas in the somata, corresponding to the cell nucleus. Scale bar = 40 μm. e Distributions of the values of the CFP/YFP ratios for single cells. The coefficient of variation, computed as ratio of SD/mean, is reported for each distribution (means ± SD, n cells: FLIIP 870 nm = 1.36 ± 0.30, n = 525; FLIIP 925 nm = 3.79 ± 1.17, n = 525; cdFLIIP 870 nm = 0.92 ± 0.06, n = 447; cdFLIIP 925 nm = 2.27 ± 0.14, n = 478). f Fraction of pixels in each cell body whose intensity is below 90% of the intensity of the mean. The significant difference in this value reflects a significant difference in the distribution of the intensity values due to the darker nuclei. Dashed lines indicate the median, 25th and 75th percentile. The p value was calculated based on Welch t test. g Baseline normalized ratiometric responses of cortical neurons expressing FLIIP (red) or cdFLIIP (black) to a bolus i.v. glucose injection. As reference, the response of cdFLIIP to an identical bolus i.v. injection of saline is shown in blue. While cdFLIIP produces a response compatible with the expected rise in glucose concentration, FLIIP does not respond to the injection. Curves are shown as mean ± SEM (N = 3 mice, n = 6 experiments). Panels b and d were generated using BioRender.

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