Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: Western blot of CD20 expression. Western blotting of different samples collected from patients carrying B malignancies. CLL chronic lymphoid leukemia, NHL non-Hodgkin lymphoma, CBL cutaneous b lymphoma. Three B cell lines were included as well as samples from healthy donors as controls. β-actin was used as a protein-loading control. CD20 expression detection was performed using a C-terminal polyclonal CD20 antibody. (*) showed additional bands excluding wt- and D393-CD20 protein signals. Chemiluminescence time exposure was 5 min

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Western Blot, Expressing

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: Characterization of new alternative CD20 transcript sequences. a Sequencing electropherograms showing junction areas resulting from alternative splicing number in brackets indicate size length deletion in nucleotides compared to the wtCD20 reference coding sequence. b Schematic alignment of newly identified sequences with wtCD20, reporting sequence deletions. Previously D393-CD20 sequence is framed. Canonical (Ca) or cryptic (Cr) donor (DS) or acceptor (AS) splice sites are reported as well as their nucleotide position from +1ATG nucleotide (in italics )

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Sequencing

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: Summary of characteristics of CD20 splice variant transcripts

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Variant Assay, Sequencing

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: Alternative CD20-transcripts, putative proteins and junction characterization. a Schematic representation of CD20 variant coding transcripts. Transmembrane domains (TM) are positioned on the linear N-ter/C-ter protein, as well as position of the main clinical anti-CD20 antibody epitopes. Rituximab ( gray line ), Ofatumumab ( blue line ) and Obinutuzumab ( red line ). Schematic representation of antibody recognition on the putative CD20 variant-proteins. AA position is provided and numbered from the first ATG (Met) start codon. b Schematic reconstitution of amino acidic (AA) junction area after splicing involving canonical ( closed boxes ) or cryptic ( open boxes ) splice. AA sequence flanking junction is provided as well as AA position

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Variant Assay, Sequencing

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: RT-PCR and RT-qPCR assays of different CD20 transcript variants. a Full-length PCR (fl-CD20) allowed amplification of all CD20 alternative transcripts. Genomic DNA (gDNA) from wild-type PG13 (ø) and PG13 transfected by wtCD20, D657-, D618-, D480-, D393-, or D177-CD20 were amplified using primers specific for the 5′ (start codon) and 3′ (stop) CD20 gene regions, common to the six transcripts. H 2 O was used as negative control (−), and the plasmid carrying the specific CD20 variant was added to the positive control (+). b CD20 variant-specific PCR was designed to amplify each alternative transcript specifically. c Proportion (in %) of each CD20 variant transcript in four different B cell lines. Means and SD calculated from seven independent experiments of RT-qPCR quantification

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Transfection, Negative Control, Plasmid Preparation, Variant Assay, Positive Control

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: Intron reintroduction within the wtCD20 coding sequence. a Schematic representation of position of alternative splice sites (ASS) in the wtCD20 coding sequence and within the constructs after reintroduction of introns 5 (CD20-int5), 3 and 6 (CD20-int3–int6), and 5 and 6 (CD20-int5–int6). ASS positions for D657 ( closed circle ), D618 ( closed star ), D480 ( closed square ), D393 (*), and D177 ( closed triangle ) are indicated. Gray and black symbols represent canonic and cryptic splice sites, respectively. b Specific RT-PCR detection of different CD20 variants in transfected HT1080 cell lines with different constructs. Plasmid was used as positive control and untransfected HT1080 cells as negative (ϕ). Raf amplification PCR was used as control for the cDNA synthesis

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Sequencing, Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Positive Control, Amplification

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: CD20 splicing quantification of EBV samples. Quantification by RT-qPCR of total (TS) and specific CD20 splice variants of a EBV-transformed B lymphoblastoid cell line, BLCL ( closed triangle ), compared to their respective PBMCs ( closed circle ); n = 6. b Infectious mononucleosis (IMN, closed rhombus , n = 4); c EBV-reactivated samples (EBV load increase >2 Log/ml. EBVr, inverted closed triangle , n = 10) after allograft compared to heathy PBMCs ( closed circle , n = 6). (*) and (**) are the results of X 2 tests with p < 0.02 and p < 0.002, respectively. CD20 transcript quantification within PBMCs reported as reference in all total or specific splicing quantification analyses. d Proportion (in %) of each CD20 transcipt variant in EBV samples in comparison with PBMC

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Quantitative RT-PCR, Transformation Assay, Variant Assay

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: RT-qPCR quantification of all CD20 splice variants within B cell malignancies. Quantification by RT-qPCR of total a or specific b , c CD20 splice variants in four B cell lines, in different B cell malignancies, compared to PBMCs from healthy donors (n = 6). c Proportion (in %) of each CD20 transcript variant in different B cell malignancies. CBCL cutaneous B cell lymphomas (n = 5), FL follicular lymphoma (n = 5), DLBCL diffuse large B cell lymphoma (n = 5), MCL mantle-cell lymphoma (n = 19), MZL marginal zone lymphoma (n = 12), MM multiple myeloma (n = 3). (*) and (**) are the results of X 2 tests with p = 0.01 and p < 0.001, respectively, compared to PBMC samples

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Quantitative RT-PCR, Variant Assay

Journal: Experimental Hematology & Oncology

Article Title: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies

doi: 10.1186/s40164-016-0036-3

Figure Lengend Snippet: RT-qPCR quantification of total or specific CD20 splice variant expression within three different CLL sample cohorts. Quantification by RT-qPCR of total a or specific b , c CD20 splice variants in three cohorts of patients: CLL patient samples collected during routine diagnosis (50.8–49.1 %, stages A–B/C, respectively) (CHU Toulouse, France, n = 70); CD19-positive B cells purified from diagnostic stages B and C (65.5 and 34.5 %, respectively); and CLL samples from elderly patients (>65 years; median, 71.2) (CLL2007-SA trial (n = 54) or from patients with relapsed stages A, B, or C (mainly stages B and C for 88.7 %) with active disease (CLL01 BOMP clinical trial, n = 70). c Proportion (in %) of each CD20 transcript variant in different CLL cohorts. (*) and (**) are the results of X 2 tests with p = 0.01 and p < 0.001, respectively, compared to PBMC samples

Article Snippet: The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies).

Techniques: Quantitative RT-PCR, Variant Assay, Expressing, Purification, Diagnostic Assay