![Figure 1. shRNAi lentiviral mini-library for targeting B-cell receptor (BCR) regulatory genes. (A) Cloning strategy for the lentiviral vector pA179.Helix. Only the relevant portions of the plasmids are shown. The pA179.Helix vector is a new version of pFUGW vector engineered at the CNIO Genomic Unit (http://www.cnio.es/es/index.asp). The pA179.Helix contains a CMV enhancer substituted for the U3 region of the 5′ LTR to maximize expression of viral RNA genomes during transient transfection.31 The pA179.Helix also contains a deletion in the U3 region of the 3′ LTR that makes the 5′ LTR of the integrated provirus transcriptionally inactive66 as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WRE) inserted downstream of GFP to increase the level of transcription.67 (B) BCR-induced programmed cell death in a group of human B-cell lymphomas. MHH-PREB-1, RAMOS RA-1, SU-DHL-6, Z-138, JEKO, REC-1, MEC-1, and DOHH2 B-NHL cells were cultured (50 000 cells/ 200 μL/ well in 96-well plates) either in medium alone (RPMI plus additives and 10% FCS) or anti-IgM treated [goat F(ab′)2 antihuman IgM (10 μg/mL) antibody]. B cells were collected at 24 hours and programmed cell death was quantified using annexin V-APC staining. Cell death was quantified by FACS analysis, followed by analysis using FlowJo software (mean ± SD). Representative flow cytometric plots are shown. Numbers on plots represent the pecentage of cells in each quadrant over the total number of cells gated. (C) Western blot for human Argonuate-2 and Dicer in total protein extracts (RIPA extraction) from Z-138, DOHH-2, and RAMOS RA-1 B cells.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/111/3/10.1182_blood-2007-09-110601/6/zh80030814510001.jpeg?Expires=1723461655&Signature=JwnIK7s1HbjKFDC4P0ALHzXy5vpSTXYaUzcNVyoXLpOUD16JmIZkJ~lEDYPnMR34AdlJ3on9ZHplA3VsHqGSVUdoBHLQlQCBtkP71RYy73S0ElmL5yKgS43f8Bb8AreHkJq9qOr2FRyA49UgltfaCQqtm8SFxqre2nJxsaF~8r2stHgJ4qLY9yN2fXVPqHAofGSnYyjPT4wkg9NECUCt-pCUakqMKB1Cp7aSRQ~JZaDw8JB-MS8DDg7hbGPwO5K1vU9YXhXgQMxvTMzPAVX91kaRpoJxRKB-y3W1PUZbl8anNnNyBikAwyQdF98cIeJOTyX0k~in1YPPRvP4w~78vQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
shRNAi lentiviral mini-library for targeting B-cell receptor (BCR) regulatory genes. (A) Cloning strategy for the lentiviral vector pA179.Helix. Only the relevant portions of the plasmids are shown. The pA179.Helix vector is a new version of pFUGW vector engineered at the CNIO Genomic Unit (http://www.cnio.es/es/index.asp). The pA179.Helix contains a CMV enhancer substituted for the U3 region of the 5′ LTR to maximize expression of viral RNA genomes during transient transfection.31 The pA179.Helix also contains a deletion in the U3 region of the 3′ LTR that makes the 5′ LTR of the integrated provirus transcriptionally inactive66 as well as the woodchuck hepatitis virus posttranscriptional regulatory element (WRE) inserted downstream of GFP to increase the level of transcription.67 (B) BCR-induced programmed cell death in a group of human B-cell lymphomas. MHH-PREB-1, RAMOS RA-1, SU-DHL-6, Z-138, JEKO, REC-1, MEC-1, and DOHH2 B-NHL cells were cultured (50 000 cells/ 200 μL/ well in 96-well plates) either in medium alone (RPMI plus additives and 10% FCS) or anti-IgM treated [goat F(ab′)2 antihuman IgM (10 μg/mL) antibody]. B cells were collected at 24 hours and programmed cell death was quantified using annexin V-APC staining. Cell death was quantified by FACS analysis, followed by analysis using FlowJo software (mean ± SD). Representative flow cytometric plots are shown. Numbers on plots represent the pecentage of cells in each quadrant over the total number of cells gated. (C) Western blot for human Argonuate-2 and Dicer in total protein extracts (RIPA extraction) from Z-138, DOHH-2, and RAMOS RA-1 B cells.
