Fig. 3: Generation of splicing-based tools using SLED vectors.
From: Cell-specific regulation of gene expression using splicing-dependent frameshifting
a Diagramatic sketch of GluA2 (Gria2) flip/flop SLED vector design (SLED.GluA2). b To validate that SLED.GluA2 reflected endogenous GluA2 flip/flop splicing patterns, we designed endogenous mRNA-specific primers to PCR amplify the GluA2 flip/flop locus. Although the mutually exclusive flip and flop exons are identical in length and highly similar in sequence, HpaI will selectively digest the flop PCR product into two fragments. SLED.GluA2, packaged into AAV9, was used to electroporate primary rat neuronal cultures and EGFPhigh/mCherrylow cells were isolated using FACS (Fig. S5). RNA was extracted from EGFPhigh/mCherrylow cells and bulk rat neuronal cultures and primers (b) were used to amplify PCR products. c HpaI incubation yielded digestion products in the EGFPhigh/mCherrylow cells, which was further confirmed using Sanger sequencing (d, Fig. S5, n = 36, ****p < 0.0001, two-tailed t-test.). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Following HpaI digestion, the undigested flip isoform remains at 392 bp, while the flop isoform yields two bands at 169 bp and 223 bp. e Primary rat neuronal cultures transduced with SLED.GluA2 show significantly different EGFP/mCherry ratios between excitatory (GAD67−) and inhibitory (GAD67+) neurons. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. ****p < 0.0001, two-tailed t-test. f Diagramatic sketch of bicistronic jRGECO1a (inhibitory neurons) and jGCaMP7b (excitatory neurons) SLED vector design (SLED.CaRPv1). g Transfection of primary rat neuronal cultures yielded divergent ratios in jGCaMP7b and RGECO1a intensities in excitatory (mDlx-Azurite−) and inhibitory (mDlx-Azurite+) neurons. Data presented represent jGCaMP7b (top row) and jRGECO1a (middle row) intensity values over a 60 s time-lapse (4 Hz) and are representative of three independent transfections of a 6-well plate. Bottom row represents a normalized representation of total Ca intensity scaled by the delta between jGCaMP7b and jRGECO1a pixel values. Individual Ca indicator traces are demonstrated in the bottom panels. For ratio calculations in panel e, n = 265 (cortex) and n = 315 (hippocampus). Scale bars = 50 μm (e) and 20 μm (g). Source data are provided as a Source Data file.
