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GLuc-ON™ Promoter Reporter Clones

Introduction Using a secreted and robust Gaussia Luciferase (GLuc) as the reporter, GeneCopoeia GLuc-ON™ promoter clones are designed for promoter analysis by detecting the real-time activities of over 20,000 human and 18,000 mouse promoters using live cell assays. Each transfection-ready promoter clone contains a 1.2-1.5 kb insert, corresponding to the 5'-flanking promoter sequence located approximately 1.5 kb upstream and up to 200 bp downstream of the transcription start site (TSS) of a specific human gene. This insert is placed upstream of the GLuc reporter gene. Since the putative cis-acting enhancer elements are expected to exist in the cloned promoter region, the promoter luciferase activity observed during the reporter assay closely resembles the actual promoter regulation of these genes within human cells. GLuc-ON promoter clones can be ordered as pre-designed or custom-built in one of our robust vector systems. Currently we offer both single-reporter and dual-reporter vector systems. The single-reporter system uses GLuc, mCherry, or GFP as the promoter reporter. The dual-reporter system uses GLuc as the promoter reporter and SEAP (secreted Alkaline Phosphatase) as the internal control for signal normalization.

mechanism of promoter reporter clones

Figure 1. How GLuc-ON promoter clones work.

 

Advantages

Live cell assays

  • Naturally secreted GLuc reporter
  • No lysis of the cells is necessary
  • Save samples, reduce variations, and simplify experiments for applications such as pulse chase analysis,etc.

Real-time study

  • Data is generated quickly
  • Closely resembles real-time activities

Dual secreted reporter system

  • Secreted GLuc and SEAP
  • Enables transfection-normalization for accurate across-sample comparison

High-throughput compatible

  • Group or pathway study compatible
  • High sample number compatible

High sensitivity

  • GLuc is 1000-fold more sensitive than firefly or Renilla luciferase

Convenience

  • All promoter clones are transfection-ready
  • Compatible with GeneCopoeia's promoter luciferase assay

 

Gaussia luciferase

GLuc-ON promoter clones use a modified GLuc (mGLuc) as the reporter gene, which generates a highly stable signal and overcomes the quick signal decay commonly observed with humanized wild type GLuc (wtGLuc).

Signal stability of mGluc and wtGlucSignal stability of mGluc and wtGluc

Figure 2. Signal stability of mGLuc (blue) and wtGLuc (red). Left: assay buffer with a stabilizer; Right: regular assay buffer (Secrete-Pair™ dual luminescence assay kit)

 

Dual-reporter system

Dual-reporter vectors are available for the GLuc-ON promoter clones. The secondary reporter, secreted Alkaline Phosphatase (SEAP), serves as an internal control. The dual-reporter system enables transfection normalization for accurate cross-sample comparison.

promoter luciferase assay   Figure 3. Normalized promoter activities in H1B1B and HEK293T cells. Dual-reporter promoter clones or controls were transfected into two cell lines in duplicates. Samples were analyzed 24 hrs (HEK293T) and 48 hrs (H1B1B) after transfection. NEG (containing non-promoter sequence) and EMPTY (no promoter in the vector) are negative controls.