Journal: Nucleic Acids Research

Article Title: Optogenetic Blue GENEs engineered into a human safe harbor locus

doi: 10.1093/nar/gkaf1461

Figure Lengend Snippet: Spatial optogenetic tissue regulation using Blue GENEs . ( A ) Architecture of the transposase-compatible R3-4-design vector with mCherry as the GOI , resulting in the production of a red fluorescent protein in blue light-illuminated cells. For abbreviations, see . ( B ) A CHO-K1-derived culture stably transfected with the vector illustrated in panel (A) was first enriched for blue light-responsive clones by illumination and cell sorting of cells with the highest 8% mean mCherry fluorescence intensity (MFI) values. Subsequently, 10 clones were randomly isolated and screened for mCherry fluorescence induction by cell culture illumination with 10 µmol m −2 s −1 blue light for 24 h following flow cytometric assessment. An arrow indicates the clone selected for further experiments (CHO-K1 #1 ). The screening experiment was performed once. ( C ) Experiment as in panel (B) but using HEK-293 cells and 19 randomly selected clones, yielding HEK-293 #7 . The screening experiment was performed once. ( D ) Live imaging time course of spatial mCherry ( mCh ) induction using CHO-K1 #1 cells over 24 h using µPatternScope for the projection of the pattern shown in the first image, encompassing variable projection intensity values ranging from 5–60 piu (projection intensity unit, see the ‘Materials and methods’ section). The rectangular shapes from left to right correspond to 5, 10, 30, and 60 piu, and the gradient ranges from 0–60 piu. See for a video, and and for a corresponding experiment using HEK-293 #7 . Scale bar, 100 µm. The experiment was performed three times with similar results. ( E ) Live imaging time course of spatial mCherry (mCh) induction in CHO-K1 cells combined with temporally offset mTagBFP2 induction using the Tet-On system. Cells were illuminated with a projected pattern using µPatternScope with a blue light projection intensity of 30 piu for 24 h. Cells were then treated with 2.5 µg ml −1 tetracycline for an additional 23 h in darkness. See for a video. Scale bar, 100 µm. The experiment was performed with unsorted cells and repeated with sorted cells (shown), yielding similar results. ( F ) Time course of optogenetic mCherry ( mCh ) induction in 3D spheroid cultures formed with HEK-293 #7 cells. A single spheroid was selected for spatial induction using µPatternScope with a blue light projection intensity of 60 piu for 72 h. The projection mask is shown in the first image. See for a video and for further analysis. Scale bar, 100 µm. The experiment was performed three times with similar results. ( G ) Spatial induction of an HEK-293 #7 spheroid using the µPatternScope, projecting a mask covering an edge of the tissue (shown in the left image). A blue light projection intensity of 20 piu was used. See for a video. Scale bar, 100 µm. The experiment was performed three times.

Article Snippet: CHO-K1 cells (DSMZ, ACC 110) were cultivated in Ham’s F12 Medium (PAN Biotech, cat. no. P04-14500) with identical supplementation.

Techniques: Plasmid Preparation, Derivative Assay, Stable Transfection, Transfection, Clone Assay, FACS, Fluorescence, Isolation, Cell Culture, Imaging