Characterization of conserved noncoding regulatory regions by flow cytometric analysis (LB66)

Spatial and temporal regulation of developmental gene transcription often involves regulatory sequences found in noncoding DNA separate from the gene’s promoter. Conservation of a noncoding region (CNR) across divergent species may indicate a regulatory region critical to basic morphogenesis. CNR-green fluorescent protein (GFP) constructs can demonstrate spatial and temporal activity; however, characterization of regulatory modules within a CNR requires quantitative, in addition to qualitative, analysis. Here we describe a novel application of flow cytometry in the characterization of CNRs. CNR-reporter constructs were generated using a limb-specific Shh regulatory region (LSSRR) linked to a minimal HSV-tk driven GFP. CNR-reporter constructs were co-electroporated with β-actin driven red fluorescent protein (RFP) into the presumptive limb of chicks. Adequately transfected limbs were analyzed using the 7-Color MACSQuant Analyzer and FlowJo software to evaluate changes in CNR activity. Data was normalized to nontransfected limbs. Limbs electroporated with the β-actin driven construct demonstrated transfection in 65.1% ± 7.9 of cells. Electroporation of the basal HSV-tk-GFP construct produced minimal fluorescence (relatively intensity 0.1 ± 0.04) in a small subset of cells (14.2% ± 4.4). Limbs electroporated with LSSRR-GFP construct demonstrated robust activity in 29.8%± 6.0 of cells, with a relative intensity of 1.2 ± 0.2. A restriction enzyme generated LSSRR fragment showed intermediate activity (relative intensity 1.0 ± 0.2) in 20% ± 8.2 of cells. Our data indicate that flow cytometric analysis can identify changes in the distribution and intensity of CNR activity, which enhances our ability to characterize CNR-related regulatory sequences.