All the attenuated Rabies plasmids, listed in Table S1 , were generated by Gibson cloning using pSAD-ΔG-F3 plasmid (Osakada et al., 2011 (link)) as starting material. Briefly, the Rabies genome was PCR amplified in 2 fragments starting from the protein to be tagged. These fragments were then mixed with the tag and/or PEST domain obtained by oligonucleotides annealing and assembled using Gibson master mix (NEB).
The lentiviral vectors used to generate the packaging cells were derived from a 3rd generation lentivirus transfer vector (gift from Michael Hastings “361 polylinker,” originally pCCL-SIN-18PPT.Pgk.EGFP-WPRE). All the lentiviral vectors were generated by Gibson assembly, opening the backbone by digestion with XbaI and KpnI and PCR amplifying the CMV promoter and the different inserts.
The lentiviral vectors used to generate the packaging cells were derived from a 3rd generation lentivirus transfer vector (gift from Michael Hastings “361 polylinker,” originally pCCL-SIN-18PPT.Pgk.EGFP-WPRE). All the lentiviral vectors were generated by Gibson assembly, opening the backbone by digestion with XbaI and KpnI and PCR amplifying the CMV promoter and the different inserts.
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