Fig. 6

Seamless vector production and targeting strategy for DUX4-mNeon reporter cassette at endogenous attH4x sites in LINE-1. a A schematic representation of λ-Int mediated in vitro intramolecular recombination between attH4X and attP4X (both present in the parental substrate vector) generating DUX4-mNeon reporter seamless vector with a recombinant attL4X junction, which can subsequently intracellularly recombine with attH4X. Successful integration of the reporter resulted in attL4X (left) and attH4X (right) recombinant sites flanking the site of integration. b PCR analysis using genomic DNA from the puromycin resistant clones (obtained with co-transfection of DUX4-mNeon reporter seamless vector and Int expressing vector Int-C3) resulted in three clones (M27, T13, T25) positive for left junction with forward primers specific to LINE-1 (F1/F2) and reverse primer (mNeon rev) and a M27 clone positive for right junction with reverse primers specific to LINE-1 (R1/R2) and forward primer (Puro fwd). Lanes: L, 1 kb DNA ladder; W, no DNA control; G, genomic DNA from parental hESCs; 27,13,25; genomic DNA from M27, T13 and T25 puromycin resistant clones. c Transfection of transgenic clones M27, T13 andT25 with DUX4 expression vector pCMV-DUX4 triggered mNeon expression in a substantial fraction of cells at day 2, indicating the functionality of the Dux4 binding sites of the integrated reporter. GFP-control, vector pCMV-GFP was used as a transfection control. d An illustration of the future application of our proposed methodology for high-throughput drug screening upon mNeon reporter activation with CMV-DUX4 plasmid. The reporter activity (mNeon expression) can be modulated depending on the compounds (inhibitors/activators) used for the screening