Plvx tre3g zsgreen1

Lentiviral production was performed in 293T cells. 293T cells were plated at ∼6 × 10⁶ cells per 100 mm dish and incubated overnight. Cells were transfected with 6 μg of pLVX-TRE3G-ZsGreen1 or pLVX-EF1α-Tet3G vectors (for the inducible cell line, Clontech) with 4 μg of pCMV-Δ8.9 and 2 μg of VSVG helper plasmids using Fugene (Promega), according to the manufacturer’s instructions. After 15 h, 293T medium was replaced with ES medium. The supernatants containing viral particles were harvested after 48 h of transfection, filtered through 0.45 μm pore-size cellulose acetate filters and supplemented with polybrene (Millipore). Infections were performed with cells plated at a density of ∼1 × 10⁶ cells for one well of 6-well plate with virus-containing supernatant supplemented with polybrene (Millipore).

Total RNA from was isolated human B cells (isolated from patients with SLE as described below) using TRIzol RNA extraction reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, cDNA was synthesized using 1 µg of total RNA and the reverse transcription system [Tiangen Biochemical Technology (Beijing) Co., Ltd.]. The FcγRIIB gene was cloned by nest PCR using Taq PCR MasterMix [Tiangen Biochemical Technology (Beijing) Co., Ltd.] and outer primers (forward primer, 5′-ATC CGC CAA GCT TTG AGA GAA GGC TGT GAC T-3′ and reverse primer, 5′-AGG GAG CTT CAG GAC TCA GGT AGA TGA CT-3′) at 58°C annealing temperature for the first PCR and inter primers (forward primer, 5′-GCC CCC GGG ACG CGT ATG GGA ATC CTG TCA TTC TTA CC-3′ and reverse primer, 5′-CTA CCC GGT AGA ATT CCT AAA TAC GGT TCT GGT CAT CAG-3′) at 56°C annealing temperature for the second PCR. The FcγRIIB gene was inserted into the lentivirus-induced expression vector pLVX-TRE3G-ZsGreen1 (Takara Bio, Inc.) with green fluorescent protein (GFP) gene to construct the mFcγRIIB lentivirus.