cas9 grna expression construct  (Addgene inc)

Journal: Blood Cancer Journal

Article Title: Identification of lenalidomide resistance pathways in myeloma and targeted resensitization using cereblon replacement, inhibition of STAT3 or targeting of IRF4

doi: 10.1038/s41408-019-0173-0

Figure Lengend Snippet: a – d Cell viability, lenalidomide response, and expression of CD147/MCT1 (from up to down) were measured in XG1LenRes ( a ) and three additional HMCLs ( b – d ) to compare control cells (vector) to the cells after depletion of CD147 by CRSPR cas9 technology with three gRNAs (#1–#3). Cell viability and drug response were set up either in the absence or presence of lenalidomide at indicated concentration, measured at day 5 after treatment. The results from a representative experiment with triplicates are shown

Article Snippet: Lentiviral constructs expressing CRISPR-associated protein 9 (Cas9) and guide RNAs (gRNAs) originally generated from Feng Zhang’s lab were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Control, Plasmid Preparation, Concentration Assay

Journal: Cancer research

Article Title: Loss of FAM46C promotes cell survival in myeloma

doi: 10.1158/0008-5472.CAN-16-3011

Figure Lengend Snippet: (A) The schematic diagram of FAM46C protein, cDNA and FAM46C gRNAs generated in this study. CRISPR-cas9 technology was used to knockout endogenous FAM46C in MM cells. (B) The expression of FAM46C in #1, #4, #5 and #6 gRNA transduced OCIMY5 cells were examined at five months after infection by immunoblotting. No FAM46C protein (both full length and short form) was detected in those cells. (C) The FAM46C deficient cells (GFP+) were admixed 30:70 with parental OCIMY5 (GFP−), followed by flow cytometry analysis of the percent of surviving GFP+ population weekly. FAM46C deleted cells have growth advantage compared with parental cells. (D–E) OCIMY5 and XG1 cells harboring control virus (NS) or two FAM46C gRNAs were treated without or with dexamethasone (5μM for XG1 and 10 μM for OCIMY5), Lenalidomide (5 μM, XG1) and Bortezomib (3.5nM, OCIMY5), and then cell viability was measured by MTT assay at day 5. The cells treated at the same condition were also harvested at day 3 for Immunoblotting assay (F). (G) XG1 cells were infected with control virus and lentivirus expressing indicated FAM46C CRISPRs. The protein lysate prepared from those infected cells at one and two months after infection was analyzed by immunoblotting. The expression of Ig light chain and BIP was decreased in FAM46C CRISPRs transduced cells.

Article Snippet: Knockout of FAM46C using CRISPR-Cas9 technology Lentiviral constructs expressing CRISPR associated protein 9 (Cas9) and guide RNAs (gRNAs) were originally generated from Feng Zhang’s lab( 13 , 14 ) and were obtained from Addgene (Cambridge, MA).

Techniques: Generated, CRISPR, Knock-Out, Expressing, Infection, Western Blot, Flow Cytometry, Control, Virus, MTT Assay