Reporter Genes Assays

Introduction of Reporter Genes Assays (RGAs)

The reporter gene unit consists of a promoter and a reporter gene. The promoter region binds transcription factors that have been activated in response to a binding event or the activation of a signaling cascade. Transcription factor binding triggers the expression of the reporter gene. When establishing an assay system, a number of considerations must be made in order to choose the optimum combination of promoter and reporter genes. The choice of the promoter will ultimately control the basal level of reporter gene activity and the degree of stimulation measured. According to different elements introduced into the cells, RGAs are mainly divided into the following categories.

  1. Introduction of reporter gene only: a specific cell line with inherent receptors is naturally responsive to a biotherapeutic, and whose corresponding transcription factors could be strongly activated or inactivated, so only the introduction of a reporter gene is required to meet the needs of detection.
  2. Co-introduction of ectopic receptor and reporter gene: no reactive cell lines or only weak reactive cell lines are responsive to some biotherapeutics due to the absence or much lower expression of inherent receptors. In this situation, co-introduction of an ectopic receptor and a reporter gene could render the cells to be responsive.
  3. Co-introduction of ectopic chimeric receptor and reporter gene: chimeric receptor containing the extracellular domain of the receptor of a biotherapeutic and the intracellular domain with clearly known signaling pathways could be stably transfected into appropriate cells. Such transgenic cells could become highly responsive to certain biotherapeutics. The system could be a valuable supplement to evaluate the ADCC activity.
  4. Introduction of split luciferase: Two distinct domains of luciferase, i.e., the large N-terminal domain and the small C-terminal domain, is fused to two signaling molecules respectively. Upon co-introduction of the two elements into an appropriate cell line, the addition of the corresponding cytokine could induce the interaction of the two signaling molecules, resulting in the complementation of the N- and C-terminals of luciferase and its functionality recovery. The split luciferase complementary assay represents a useful option for RGA in the bioactivity determination of biotherapeutics.

Overview of RGA approaches. Fig 1. Overview of RGA approaches. (Miraglia, 2011)

Application of Reporter Genes Assays

Although the state-of-the-art sophisticated physicochemical assays could provide a great amount of structure information, bioassays are still irreplaceable in determining biological activity and the indication of proper high-order structures of biopharmaceuticals. The development of biological assays for products is essential for their advancement as therapeutic products. By engineering appropriate cell lines stably transfected with plasmids consisting of luciferase genes under the control of regulatory elements that represent the signaling pathways related to individual biopharmaceuticals, RGAs provide an alternative to other traditional cell-based biological assays, facilitating the R&D and quality control of biopharmaceuticals. RGAs would provide an MOA-based, less variable, more robust technique for the bioactivity determination of biopharmaceuticals. Along with the rapid growth of biopharmaceuticals with various new targets, RGAs have been widely applied in different stages of biopharmaceuticals, such as drug screening, product release, stability evaluation, and comparability study.

With superior assay performance including simple operation, short assay time, small variation, high sensitivity, and accuracy, RGAs are typically applied in supplementing the following bioassays:

Reporter gene assays for bioactivity determination of biotherapeutics. Fig. 2. Reporter gene assays for bioactivity determination of biotherapeutics. (Wang, 2020)

Reporter genes assays have important status in drug discovery methods. With extensive experience and professional experts, Creative Biolabs has established a comprehensive technology platform offering various drug discovery-relevant services to global clients. If you are looking for a partner in drug discovery, or you have any questions about our services, please don't hesitate to contact us for more information.

References

  1. Miraglia, L. J.; et al. Seeing the light: luminescent reporter gene assays. Comb Chem High Throughput Screen. 2011, 14(8): 648-57.
  2. Wang, L.; et al. Development of reporter gene assays to determine the bioactivity of biopharmaceuticals. Biotechnol Adv. 2020, 39: 107466.
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