Fig. 1: Summary of dFLASH LV-REPORT construction, utility, and validation.

a The dFLASH system utilises the lentiviral LV-REPORT construct, consisting of a cis-element multiple cloning site (ECS) for enhancer insertion (flanked by ClaI and AscI restriction enzyme sites), followed by a minimal (min) promoter that drives a transcription factor (TF) dependent cassette that encodes three separate expression markers; a nuclear Tomato fluorescent protein with a 3x C-terminal nuclear localisation signal (NLS), Herpes Simplex Virus Thymidine Kinase (HSVtK) for negative selection and, separated by a 2A self-cleaving peptide (2A), Neomycin resistance gene (Neo) for positive selection. This is followed by a downstream constitutive promoter (PGK/CMV) that drives an independent cassette encoding EGFP with a 3x N-terminal NLS and, separated by a 2A peptide, a Hygromycin (Hygro) resistance selection marker. This construct is flanked either side by long terminal repeats (LTR) for lentiviral mediated insertion. b This design allows for initial identification of the EGFP fluorescent protein in nuclei, independent of TF-dependent signal. Expression of the Tomato fluorescent protein is highly upregulated in a signal-dependent manner. Images shown are monoclonal HEK293T dFLASH-HIF cells. Populations were treated for 48 h ± DMOG to induce HIF-1α and were imaged by HCI. Data representative of n = 3 independent experiments. Scale bar = 100 μM. c This system can be adapted to a range of different applications. This includes (clockwise) flow cytometry, arrayed screening in a high throughput setting with high content imaging, isolation of highly responsive clones or single cells from a heterogenous population or temporal imaging of pooled or individual cells over time. Created in BioRender. Peet, D. (2025) https://BioRender.com/b38b268.