To accommodate changing environment, cell alters gene expression program. This adaptive response is mediated by intracellular network of regulatory pathways that relay to genomic DNA the signals that modulate gene expression.

The signaling pathways communicate with DNA by using transcription factors (TFs), proteins that bind specific sequences within target genes. By assessing TFs’ activities one can characterize the functional status of cellular gene regulatory network.

Attagene has developed and patented technology, termed FACTORIAL™, that enables quantitative assessment of activities of multiple TFs within cell.


Quantitative assessment of multiple TFs in a single assay

At the core of FACTORIAL™ is a set of reporter DNA constructs called reporter transcription units (RTUs). Each RTU has a TF-specific promoter that regulates transcription of adjacent reporter sequence. When a library of several RTUs is introduced into cell, the spectrum of reporter RNAs mirrors the activities of the evaluated TFs.

To quantify reporter RNAs we have developed an unconventional multiplexed detection approach. A principal and distinct feature of FACTORIAL™ is that all RTUs have identical reporter sequences that differ only by the position of an endonuclease recognition site (HpaI).

To assess RTU activities, reporter RNAs are amplified by RT-PCR, labeled with a fluorescent label, and digested by HpaI. The digest produces a spectrum of labeled DNA fragments of different lengths that are resolved by capillary electrophoresis (CE) and detected as separate fluorescent peaks. The intensities of those peaks are proportional to activities of evaluated TFs.

Extraordinary robustness, reproducibility and accuracy

Because all RTUs produce nearly identical RNAs, these reporter messages are equally susceptible to broad variations in experimental and detection conditions, such as degradation, amplification, and thus their relative concentrations remain unaffected. From this homogeneity stems an amazing robustness and reproducibility {link to Nat Met–figure 3} of assessments.

assessing compounds’ impact on multiple TF families

Another name for the above described FACTORIAL™ is cis-FACTORIAL, because RTU transcription is controlled by a cis-regulating element (promoter). The specificity of a RTU is determined by the presence of a TF binding site in the promoter. Importantly, as all members of TF family recognize the same binding sequence, cis-FACTORIAL™ evaluates activities of TF families.

Attagene offers two different cis-FACTORIAL™ assays, cis-FACTORIAL™-1 and cis-FACTORIAL™-2 that encompass different sets of TF families. Combined, the cis-FACTORIAL assays enable assessing over 90 gene regulatory pathways.

profiling agonist/antagonist properties of compounds across the panel of nuclear receptors

Trans- FACTORIAL™ is an embodiment of the FACTORIAL™ platform that is designed for assessing agonist/antagonist properties of compounds across multiple NRs.

The trans- FACTORIAL™ comprises a library of one-hybrid reporter constructs (trans-RTUs). A trans-RTU expresses a chimera GAL4-NR protein that regulates transcription of a reporter sequence. The presence of agonists/antagonists of NR alters the transactivation function of Gal4-NR and modulates reporter transcription.

The individual trans-RTUs have identical reporter sequences that are tagged with the HpaI site. The homogeneity of the library affords remarkable reproducibility and accuracy of assessments.

Our first trans-FACTORIAL™ assay was trans-FACTORIAL™-1 that encompassed 25 NRs and was used for the evaluation of ToxCast compounds. We now offer the comprehensive trans-FACTORIAL trans-FACTORIAL™ assay that covers all 48 human NRs.