Pcep4

Δ2GGCX lacking exon 2 was constructed by overlap PCR using a wild
type carboxylase cDNA as template [16 (link)]
and primers A-D (Supplemental
Table). The C-terminus contained a C-terminal Ala linker followed by
a FLAG tag (AAADYKDDDDK). The PCR product was cloned into the TOPO vector
(pCR4-TOPO, Thermo Fisher) and then sequenced to verify the mutation. A BamH I
fragment encoding Δ2GGCX_flag was then subcloned into pBacPAK8
(Clontech) for expression in SF21 insect cells. Δ2GGCX_flag was
also subcloned into pCMV6-AC (Origene) for expression in mammalian cells, in
parallel with wild type carboxylase_flag. An untagged Δ2GGCX
variant was also constructed for expression in mammalian cells. BamH I-EcoR I
and EcoR I-BamH I fragments containing the N-terminal exon 2 deletion mutation
and C-terminal wild type carboxylase sequences were ligated into the BamHI site
of pCEP4 (Thermo Fisher), and the carboxylase cDNA was then sequenced.

h38C2_Arg IgG1 was cloned from previously reported HC and LC expression vectors encoding anti-HER2-DVD-IgG1.^{9 (link)} Removal of the outer variable domain (anti-HER2) and the Lys99Arg mutation were done by overlap extension PCR. The modified expression cassettes were XhoI/NheI-transferred to mammalian expression vector pCEP4 (Thermo Fisher Scientific). The resulting HC and LC expression vectors were transiently transfected into Expi293F cells (Thermo Fisher Scientific) using Expi293 reagents (Thermo Fisher Scientific) and following the manufacturer’s instructions. After 6 days, cells and debris were pelleted and the medium was collected. h38C2_Arg IgG1 was purified from the collected medium using a 1-mL Protein A HP HiTrap column (GE Healthcare). The antibody concentration was determined by measuring the absorbance at 280 nm, and the purity of the antibody was confirmed by SDS-PAGE followed by Coomassie blue staining (Supplementary Figure S3). Control antibody h38C2 IgG1 (= h38C2_Lys IgG1) was a gift from the laboratory of Dr. Carlos F. Barbas III (The Scripps Research Institute).

Sequences encoding light chain (V_κ-C_κ) and heavy chain fragment (V_H-C_H1) of h38C2 Fab containing a Lys99Cys (K99C) and Lys99Tyr (K99Y) mutation in V_H and an N-terminal human CD5 signal peptide (MPMGSLQPLATLYLLGMLVASVLA) were cloned into mammalian expression vector pCEP4 (Thermo Fisher) via NheI/XhoI (New England Biolabs). Following maxipreparation (Qiagen), plasmids encoding V_κ-C_κ and V_H-C_H1 were co-transfected into Expi293F cells (grown to a density of 3 ⅹ 10⁶ cells/mL in 300 mL) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher). After continued culturing at 37°C, 5% CO₂ for 5 days, the culture supernatant was collected and subjected to purification by affinity chromatography using a 1-mL HiTrap KappaSelect column (Cytiva) on an ÄKTA FPLC instrument (Cytiva). The Pierce BCA Protein Assay Kit (Thermo Fisher) was used to determine the yield of Fab (~15 mg/L). Further purification of the Fab was done by size-exclusion chromatography (SEC) using a Superdex 200 10/300 GL column (Cytiva) on an ÄKTA FPLC instrument. Following SEC, Fab peak fractions were concentrated using an Amicon Ultra 0.5-mL Centrifugal Filter (MilliporeSigma) and brought into 0.1 M sodium acetate (pH 5.5).

The sequences encoding for E-301 and E-301-LOF containing Salmonella typhimurium sialidase were cloned into the mammalian expression vector pCEP4 (Thermo Fisher Scientific). Human embryonic kidney 293T cells were then transiently transfected with the DNA constructs using the Expi293 system and following standard protocols according to the manufacturer’s instructions (Thermo Fisher Scientific). Purification of E-301 and E-301 LOF was performed directly from transfection harvests using a HiTrap Protein A affinity column (GE Healthcare) and eluted with 1 M arginine (pH 3.9). Anion-exchange chromatography was used as a secondary purification method, and the final product was dialyzed into PBS (pH 7.4). The biochemical characterization of E-301 and E-301 LOF, including purity, HER2 binding affinity, and enzymatic activity, was conducted as described previously (25 ).

To generate the TVA-G monosynaptic tracing allele, cDNA encoding TVA and Rabies-G was excised from pBOB-synP-HTB (Addgene plasmid #30195; (Miyamichi et al., 2011 (link)) and subcloned into a custom pminiTol2 expression construct (Addgene plasmid #31829; (Balciunas et al., 2006 ) harboring a PGK promoter (pPL451; (Liu et al., 2003 (link)), hygromycin resistance cassette (pCEP4, ThermoFisher Scientific) and BGH poly-A sequence (pcdDNA3.1, Invitrogen) using high-fideity Phusion polymerase (New England Biolabs) and In-Fusion HD Cloning System (Clontech). The TVA-G Tol2 transfer vector was nucleofected into Hb9::GFP ESCs with pCMV-Tol2 transposase (Addgene plasmid #31823, (Balciunas et al., 2006 ) using Amaxa Nucleofector Kit for Neural Stem Cells (Lonza), followed by hygromycin selection (Sigma-Aldrich). Selected clones were differentiated using standard RA/SAG protocol to generate MNs. A red fluorescent protein variant (dsRed) of SADB19ΔG RABV was added at low titer to Day 6 EBs to assess efficiency of viral transfer.