luciferase-based gene reporter assay  (Promega)

Journal: Frontiers in Immunology

Article Title: Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters

doi: 10.3389/fimmu.2023.1247544

Figure Lengend Snippet: Methylation status and function analysis of the promoter region of nAChR . (A) Whole-genome bisulfite sequencing (WGBS) was used to detect methylation sites within the DMG promoter region. The horizontal axis represents the position of the promoter region of the gene in the chromosome, and the vertical axis shows the methylation level of each methylation site. The arrow direction indicates the gene’s chain orientation, where the right represents the positive chain, and the left represents the negative chain. The dashed line represents the methylation site in this region to be validated by another technique. (B) Bisulfite sequencing PCR was used to determine the methylation status of promoter-associated CpG residues in nAChR genes. The filled (black) circles correspond to methylated cytosine, the unfilled (white) circles correspond to unmethylated cytosine, and the small vertical lines without a circle correspond to the non-CpG position where there is a CpG in the genomic sequence. Each box corresponds to one CpG position in the genomic sequence. The colored bars summarize the methylation states of all sequences at that position. (C) Luciferase reporter constructs were used to analyze promoter expression in HEK-293T cells. Line 1 shows the location and size of promoter fragment relative to the transcription start site, as well as the location of methylation sites within the promoter region in pearl oysters. Line 2 indicates the predicted transcription factor in the dense methylation region of the promoter. Line 3, pGL3-gene promoter-2, which lacked the dense methylation region of the promoter, and Line 4, pGL3-gene promoter-1, which contained this region. The results of the luciferase reporter assay are presented in columns, with the mean and standard deviation (bars) shown. The data are representative of three independent experiments.

Article Snippet: The PCR products were then inserted into the pGL3-base vector (Promega, Madison, WI, USA) containing the luciferase reporter gene in accordance with the manufacturer’s instructions.

Techniques: Methylation, Methylation Sequencing, Sequencing, Luciferase, Construct, Expressing, Reporter Assay, Standard Deviation

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The Y150-dependence of gene activation of Jurkat cells: in presence of Jurkat-CD3-NFAT-RE-Luc cells only (in Green), or in presence of Jurkat-CD38 − -CD3-NFAT-RE-Luc cells only (Jurkat-4-3-F11, in Brown), or in presence of both Jurkat-CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Red), or in presence of both Jurkat- CD38 − -CD3-NFAT-RE-Luc cells and NCI-H929 cells (in Blue). In the reporter gene assay, Jurkat cells (effector cells) and NCI-H929 (target cells) were in a ratio of 1.5:1 (E:T).

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Activation Assay, Reporter Gene Assay

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: Validation of reporter gene assay. (A) Specificity assessment of the assay. Y150 or a nonspecific antibody was incubated with both NCI-H929 cells and Jurkat T cell line 4-3-F11, and the RLU signals were measured. Y150 sample stressed under oxidization with 4% hydrogen peroxide was tested and compared with Y150 reference sample. (B) Analysis of linearity of the assay was conducted by comparing the measured potency and expected potency. Each point represents the mean of six independent experiments.

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Incubation

Journal: Antibody Therapeutics

Article Title: Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

doi: 10.1093/abt/tbab022

Figure Lengend Snippet: The correlations of reporter gene assay results with those from cytotoxicity assay and sandwich ELISA assay

Article Snippet: Promega Corporation developed a cell-based luciferase reporter gene assay to monitor the Jurkat T cell activation through binding to CD3 on the Jurkat T cells engineered with an nuclear factor of activated T cells response element (NFAT-RE) driving luciferase expression [ , ].

Techniques: Reporter Gene Assay, Cytotoxicity Assay, Sandwich ELISA