Pmxs puro retroviral vector

The pMXs-Puro Retroviral Vector (Cell Biolabs Inc., San Diego, CA, USA) was used to generate cell lines with stable G protein expression. The G genes of each genotype were inserted in the multiple cloning site of the pMXs plasmid, and retroviral vectors were produced using the Platinum-GP Retroviral Packaging Cell Line (Cell Biolabs) and pMD2.G according to the manufacturer’s instructions for the expression of vesicular stomatitis virus G protein. Three days post transfection, the supernatants were collected as the retrovirus-containing solution and added to each cell line (OL, QT6, DF-1, and 3T3 cells). Cells permanently inoculated with retrovirus were selected in medium containing puromycin (8 µg/mL).

FLAG-tagged G3BP1 expression plasmid (pFLAG-G3BP1) and its mutants were described previously^{20 (link)}. The pMXs-FLAG-G3BP1-Puro was a G3BP1 retroviral vector plasmid constructed by inserting a FLAG-G3BP1 DNA sequence prepared from the pFLAG-G3BP1 plasmid by polymerase chain reaction into a multicloning site of the pMXs-Puro retroviral vector (Cell Biolabs, Inc., San Diego, CA, USA). The HA-tagged USP10 (HA-USP10) expression plasmid was described previously^{14 (link)}. pMD.G is the expression vector of the envelope glycoprotein (G protein) of vesicular stomatitis virus and a kind gift from Dr. Didier Trono (Swiss Federal Institute of Technology in Lausanne, Switzerland). GFP-CFTR-ΔF508 plasmid was a gift from Dr. Ron Kopito (Stanford University, Palo Alto, CA, USA). α-synuclein plasmid was obtained from Dr. Masato Hasegawa (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). YFP-CL1 plasmid was a gift from Nico Dantuma. The six-His-ubiquitin plasmid was provided by Dr. Dirk Bohmann (University of Rochester Medical Center, Rochester, NY, USA).

We used the nucleotide sequences of NPC1 genes derived from SuBK12-08, YubFKT1, BKT1, FBKT1, ZFBK13-76E, ZFBK11-97, ZFBK15-137RA, and DemKT1, which were determined previously (GenBank accession numbers; LC462997, LC462271, LC462998, LC462999, LC462993, LC462994, LC462995, and LC462996, respectively [20 (link)]). Total RNA was extracted from SuBK12-08 cells using ISOGEN (Nippongene) and mRNA was reverse transcribed with Superscript IV (Invitrogen). The NPC1 gene of SuBK12-08 was amplified with KOD One and inserted into a pSP72 (Promega, Madison, WI, USA) plasmid vector. After sequence confirmation, domain C of the NPC1 gene fragment derived from SuBK12-08 cells (amino acid residues 377–624 [373–620 in HEK293T NPC1 numbering]) and the NPC1 gene fragment derived from HEK293T cells (amino acid residues 1–372 and 621–1279) were amplified with KOD One using specific primers with the HA tag sequence. Then these NPC1 gene fragments were inserted into the pMXs-puro retroviral vector (Cell Biolabs. San Diego, CA, USA) to construct pMXs-chimeric HEK293T/SuBK12-08 NPC1. An In-Fusion cloning kit (BD Clontech) was used to construct the retroviral vectors carrying NPC1 genes. The plasmids of mutant NPC1 genes were constructed by site-directed mutagenesis with KOD One. After sequence confirmation, these mutant genes were inserted into the retroviral vector.

Murine CAR-T cells were produced as previously described [22 (link)]. Briefly, a murine leukaemia virus-derived pMXs-Puro Retroviral Vector (Cell Biolabs, San Diego, CA, USA) was used as a plasmid for Rv production. The Rv packaging CAR gene was produced by transfecting Plat-E cells with the pMXs-Puro/CAR. The culture supernatant of Plat-E cells obtained 48 h later was filtered through a 0.45 μm filter and used as an Rv solution for gene transfer. Murine T cells were activated by incubation with an anti-CD3ε mAb (clone 145-2C11, Bioxcell, West Lebanon, NH, USA) and anti-CD28 mAb (clone 37.51, Bioxcell, West Lebanon, NH, USA) and then transduced with Rv-bound Retronectin (Takara Bio, Kusatsu, Japan) under anti-CD3ε/CD28 mAbs stimulation. The gene-transduced cells were cultured in cRPMI medium, supplemented with 5 μg/mL puromycin. We defined the end of the 24 h culture as the end of the gene transfer operation on the 0th day (day 0).

Human FLT3^wt (NM_004119) and FLT3-ITD were N-terminally tagged with flag and cloned into the pMXs-Puro retroviral vector (Cell Biolabs). The ITD sequence of FLT3-ITD was derived from MOLM-13 cells [33 (link)]. FLT3LG (a. a. 1-181, NM_001204502) encoding the soluble form of FL was cloned into the pMXs-Puro retroviral vector.