Skip to main content
. 2022 Aug 30;11:e79848. doi: 10.7554/eLife.79848

Figure 1. A new viral packaging system for fast, clean and high-throughput production of RVdG-CVS-N2c vectors.

(A–C) A schematic representation of the experimental workflow for achieving cell type-specific trans-synaptic retrograde labeling using G-deleted, envA-pseudotyped rabies viral vectors: Genetic dissection of cre+ neurons (blue) for conditional expression of TVA and G (A); targeting of RVdGenvA particles to labeled neurons for expression of a gene of interest (GOI), (B) and subsequent propagation of native-coat RVdG particles from starter cells to their presynaptic partners (C). (D) A schematic representation of the three different cell lines designed for rescue (HEK-GT), pseudotyping and amplification (BHK-eT) and titration (HEK-TVA) of RVdG viral particles, alongside the transgenes used to generate them. (E) Schematic representation of the production process and timeline. L,P and N represent the plasmids encoding the corresponding rabies genes and V represents the vector plasmid. (F) Quantification of the time course for the rescue stage, starting at day 4 after transfection of viral plasmids. (G) Quantification of the time course for amplification of pseudotyped particles, starting at day 1 after transduction with native-coat particles. (H) Quantification of the average titer of concentrated pseudotyped stock from 19 individual productions, with comparison to the titer of a representative production of RVdg-CVS-N2c virus, produced using N2a cells (magenta). Lines represent titers of individual productions. Data in F and G represents the average and SEM of three individual and independent measurements.

Figure 1—source data 1. Quantification of RVdG propagation rates and viral titers.
Tables for Figure 1F and G show the fractions of labeled cells obtained using FACS analysis at the indicated days post transduction and table for Figure 1H details viral titers for 37 individual productions, along with the identity of each vector and the raw data used to calculate the titer (colored columns to the left).

Figure 1.

Figure 1—figure supplement 1. Efficiency of stable cell selection using antibiotic resistance genes.

Figure 1—figure supplement 1.

(A and B) HEK-GT (A) and BHK-eT (B) cells (bright-field illumination shown in cyan) were mixed with HEK293 or BHK-21 cells, respectively, stably expressing tdTomato only (red). Representative images show the gradual removal of tdTomato+ cells following exposure to either of the antibiotics Puromycin, blasticidin, or neomycin used in the generation of the stable cells. Scale bars represent 100 µm. (C) FACS-assisted quantification of the fraction of tdTomato+ cells shows the time course for their removal from the antibiotics-exposed cultures.

Figure 1—figure supplement 2. Leak expression of envA-pseudotyped particles can occur around damaged tissue.

Figure 1—figure supplement 2.

(A) Illustration of the injection scheme designed to reveal the extent of leak expression following injection of envA-pseudotyped RVcG-CVS-N2c particles produced in BHK-eT cells. (B) Representative confocal image of a coronal section (left) shows extensive labeling of low-titer N2c-tdTomato vectors (magenta) from the hemisphere previously injected AAV-DIO-EF1a-TVA-2A-N2cG but only a small number of cells labeled with N2c-EGFP (green, left) which was injected into the contralateral hemisphere at a titer 500-fold higher. DAPI signal is labeled blue. (C) Protocol for the experiment in organotypic hippocampal slice cultures, designed to assess the contribution of tissue damage to non-specific labeling by envA-pseudotyped particles. (D and E) Images from three separate cultures transduced with envA-pseudotyped CVS-N2c-tdTomato vectors (red), either immediately (D) or 1 hr after the incision of the tissue (E). The substantially higher number of labeled cells in the cultures shown in (D) suggests that non-specific labeling from envA-pseudotyped particles can occur around injection sites when high-titer virus is applied.

Figure 1—figure supplement 3. Effective retrograde labeling with oG coated CVS-N2c particles.

Figure 1—figure supplement 3.

(A) Illustration of the injection scheme for hippocampus-specific retrograde labeling with native coat RVdGoGCVS-N2c-EGFP particles. (B) Representative coronal (left) and sagittal (right) images demonstrating specific and robust retrograde labeling of cortical and subcortical regions projecting to the hippocampus. LEC – Lateral entorhinal cortex; DBB – Diagonal band of Broca; Re – Nucleus reuniuns of thalamus; SuM – Supramammilary Nucleus; MnR – Median nucleus Raphe. 4′,6-diamidino-2-phenylindole (DAPI) signal is labeled blue.