Molecular cloning and AAV production
The original Ribo-STAMP construct consisted of a Tet-On hRPS2–APOBEC lentiviral expression vector, with a human phosphoglycerate kinase (PGK) promoter driving the expression of rtTA and pac, designed for generation of stable human cell lines18 (Fig. 1b). ORFs of Ribo-STAMP variants were first cloned into a plasmid derived from the pLIX_403 vector (a gift from D. Root; Addgene plasmid no. 41395) containing a P2A-mRuby fluorescent protein sequence in-frame with the fusion protein. The RPS2–APOBEC fusion sequence was taken from the pLIX403_Capture1_RPS2_APOBEC_HA_P2A_mRuby construct, as used previously18. Following the EGFP-tagging methodology of the TRAP system61, the ORF isoform 1 of the mouse Rpl10a was fused to the C terminus of APOBEC1. A similar strategy was adopted for the Rpl22–APOBEC fusion sequence whereby the mouse Rpl22 ORF was fused to the N terminus of APOBEC to mimic the haemagglutinin-tagging strategy of RiboTag62. Both fusion sequences were generated as synthetic gBlocks (Integrated DNA Technologies) and cloned into the pLIX403_Capture1_APOBEC_HA_P2A_mRuby parental backbone using Gibson assembly. The Tet-On Ribo-STAMP constructs used in Figs. 1 and 2 were cloned into a mammalian gene expression AAV vector (VectorBuilder) containing a truncated woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation signal (BGH polyA), using Gibson assembly. The tetracycline-responsive promoter element, followed by Ribo-STAMP variant ORFs, was polymerase chain reaction (PCR)-amplified from the previously described plasmids. The hSyn promoter was PCR-amplified from pAAV.Syn.GCaMP6s.WPRE.SV40 (a gift from D. Kim; Addgene plasmid no. 100843), and the rtTA was PCR-amplified from the pLIX_403 vector. Ribo-STAMP constructs used in Figs. 1 and 2 were cloned into a mammalian gene expression AAV vector (VectorBuilder). Constitutive RPS2-STAMP used for in vivo experiments was assembled by VectorBuilder. The construct was cloned into a mammalian gene expression AAV vector (VectorBuilder) containing an EF1A promoter, the RPS2_APOBEC ORF containing an haemagglutinin tag as above and fused with TdTomato through a P2A peptide, followed by a truncated WPRE and BGH polyA. AAVs were prepared following Challis et al.63 using the PHP.eB capsid for packaging.
Animals
C57BL/6J mice were group-housed on a 12-h light–dark cycle and fed a standard rodent chow diet. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Scripps Research Institute and were in accordance with the guidelines from the National Institutes of Health (NIH).
Primary neuron culture, treatments, infections and viability test
Cortices were dissected from E18 C57BL/6J embryos in Leibovitz’s L-15 Medium (Life Technologies) and incubated in TrypLE Express (Life Technologies) at 37 °C for 4 min. Neurons were mechanically dissociated and plated on poly-d-lysine-hydrobromide-coated six-well plates (600,000 cells per well) or on 12-mm coverslips in 24-well plates (100,000 cells per well). Neurons were maintained in a humidified environment at 37 °C and 5% CO 2 in neurobasal medium (Life Technologies) supplemented with B27 Plus Supplement (Life Technologies) and GlutaMAX (Life Technologies). At day in vitro (DIV) 3, floxuridine was added to the medium to limit the growth of dividing cells. DIV 6 neurons were infected with 1 × 1013 (24-well plates) or 4 × 1013 (six-well plates) viral genomes ml−1 of inducible Ribo-STAMP AAV virions. Ribo-STAMP expression was induced with 1 µg ml−1 Dox for 48 h before collection at DIV 14. BDNF (100 ng ml−1) was applied 15 min and 60 min before collection64,65. Anisomycin (40 µM) was applied 30 min before collection. For Puro-PLA experiments, neurons were treated with puromycin (10 μm) for 15 min before collection. Cell viability assay was performed using the Cell Counting Kit Assay-8 (MilliporeSigma).
Proximity ligation assay
Proximity ligation assay (PLA)65 was performed using the Duolink kit (Sigma). After puromycin treatment, neurons were washed in PBS, fixed for 10 min in 4% paraformaldehyde (PFA) and washed again in PBS. Fixed neurons were permeabilized with 0.1% Triton X-100 in PBS-CM (PBS (pH 7.4), 1 mM MgCl 2 and 0.1 mM CaCl 2 ) for 10 min. All reactions were incubated in a preheated humidity chamber. After a brief wash with PBS-CM, each sample was blocked with Duolink Blocking Solution for 1 h at 37 °C and then incubated with anti-puromycin and protein-of-interest primary antibodies diluted in Duolink Antibody Diluent overnight at 4 °C. After two washes in wash buffer A, PLA rabbit plus and mouse minus probes were applied at a 1:5 dilution in Duolink Antibody Diluent for 1 h at 37 °C. After washes, ligation and amplification reactions were performed using Duolink Fluorescent Detection Reagent in FarRed. The cells were incubated in the ligation reaction for 30 min at 37 °C, washed with wash buffer A and incubated in the amplification reaction for 100 min at 37 °C. Following the final washes with 1× and 0.01× wash buffer B, cells were postfixed for 10 min with 4% PFA, washed in PBS and prepared for immunocytochemistry.
Immunocytochemistry
PFA-fixed cells were permeabilized with 0.1% Trixon-X 100 in PBS for 10 min at room temperature, washed in PBS and blocked with 10% normal goat serum (NGS) in PBS for 1 h at room temperature. Cells were incubated with primary antibodies at desired concentrations in 1% NGS in PBS overnight at 4 °C. After three washes with PBS, cells were incubated with secondary antibodies at 1:500 dilution in 1% NGS in PBS for 2 h at room temperature. After three washes with PBS, coverslips were mounted onto microscope slides (Thermo Fisher Scientific) with ProLong Diamond Antifade Mountant (Life Technologies) and imaged.
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