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. 2022 Aug 30;11:e79848. doi: 10.7554/eLife.79848

Figure 6. An extended suite of RVdG-CVS-N2c vectors for bicistronic expression of fluorescent markers and optogenetic effectors.

(A) Schematic illustration of the vector sequence, designed to drive independent bicistronic expression of a nuclear-localized EGFP (nl.EGFP) alongside tdTomato, using the N-P linker sequence (top). Representative confocal images of the HC (bottom right) and EC (bottom left) following retrograde labeling from the DG, demonstrate the differential localization, indicating effective separation of the fluorophores. (B) Schematic diagram of a bicistronic nl.EGFP +SypGFP CVS-N2c vector (top) used for retrograde labeling from the DG (right panels) and representative confocal images demonstrating dual nuclear and synaptic localization of EGFP in the dentate granular and molecular layer (top right image) and purely synaptic localization at the mossy fibers terminals (bottom right image). (C) Schematic diagram of a bicistronic tdTomato +oChIEF CVS-N2c vector (top) used for retrograde labeling from the DG, and a representative image of a biocytin-filled neuron (white) in MEC-2 (bottom left) along with representative traces from 10 overlaid recordings at different frequencies (bottom right). (D) Summary plots of the action potential success rate for recordings made 6–7 days after introduction of RVdG (left) and their resting membrane potential at the time of recording (right) demonstrate the light responsiveness and physiological condition of the labeled neurons. (E) Representative confocal images (top) of DGCs (left), DG molecular layer interneurons (center) and CA3 pyramidal neurons (right) and their synaptic responses to optogenetic activation of the perforant path (bottom) following retrograde labeling from the dorsal DG with the bicistronic CVS-N2c-tdTomato-oChIEF vector.

Figure 6—source data 1. Firing rate and membrane potential properties of neurons transduced with CVS-N2c-tdTomato-ChIEF, 7 days post transduction.
The table details the firing properties of 6 cells to stimulation frequencies of 25, 50, and 100 Hz and 12 additional cells to stimulation of 25 Hz only. The average resting membrane potential for each cell is shown in the rightmost column.

Figure 6.

Figure 6—figure supplement 1. Additional properties of bicistronic RVdG-CVS-N2c vectors.

Figure 6—figure supplement 1.

(A and B) FACS analysis of fluorescence intensity in HEK-TVA cells transduced with RVdGenvA-CVS-N2c-nl.EGFP-tdTomato (A) or HEK293 cells transduced with 10 X concentration of the same vector (B). Five different clusters of labeled cells are highlighted in (A), likely representing: (1) tdTomato-/nl.EGFP- Non-transduced cells; (2) tdTomato-/nl.EGFP+ Cells with a putative null mutation in the tdTomato gene; (3) tdTomatolow/nl.EGFP- putatively undergoing mitosis and lacking a defined nucleus; (4) tdTomato+/nl.EGFP- with a putative null mutation in the EGFP gene; (5) tdTomato+/nl.EGFP+ with no putative mutations. (C) Quantification of fluorescence intensity for three different RVdG-CVS-N2c vectors, in which tdTomato is expressed alone, or in a bicistronic vector in either the first (tdTomato-FlpO) or second (nl.EGFP-tdTomato) position shows no substantial differences in tdTomato fluorescence intensity. (D) A representative, high-magnification confocal image of the granule cell layer of the DG, following targeting of the RVdGenvA-CVS-N2c-nl.EGFP-tdTomato vector, showing a cell expressing a soluble EGFP, which is likely the result of a mutation in the nuclear localization signal.