Fig. 2: Efficient targeted integration of α-PD-1 mAb into human primary B cells. | Cell Death & Disease

Fig. 2: Efficient targeted integration of α-PD-1 mAb into human primary B cells.

From: Engineering of α-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

Fig. 2

a Comparison of transduction rates between BaEVTR and VSVG pseudotyped IDLVs. An incubation of the freshly pre-stimulated B cells with BaEVTR or VSVG pseudotyped GFP-encoding IDLVs (MOI of 10, based on titering via flow cytometry for GFP expression) was conducted for 48 h at 37 °C, followed by FACS analysis for detection of GFP+ cells. Pre-stimulated B cells without transduction were used as the control. b A wave of transgene expression was observed at human primary B cells transduced with BaEVTR pseudotyped IDLV. GFP expression was estimated by FACS at 24 h, day 7 or day 14 post transduction. c Detection of BaEVTR pseudotyped ICLVs and IDLVs integration at 24 h or day 14 post transduction with a modified Alu-LTR nested–PCR protocol. Results are presented as mean ± SEM, n = 3. d Schematic representation of the human primary B cells engineering protocol by infection of dual-IDLVs. e Representative flow cytometric analysis for integrated CD90 expression 5 days post infection as indicated in d. Pre-stimulated B cells with only the donor IDLV were used as the control. f The relative knock-in efficiency in human primary B cells transduced with dual-IDLVs are shown. Data are from three independent experiments (means ± SEM), ***P < 0.001; two-tailed Student’s t test (f) was used.

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