Targeting constructs were generated using a combined gene synthesis (GenScript) and molecular cloning approach. ChR2(H134R)-tdTomato was synthesized based on the hChR2 sequence 26 (link), and ChR2(H134R)-EYFP was synthesized based on the ChR2 sequence 3 (link). Both fragments, as well as the ss-Arch-EGFP-ER2 6 (link) and the eNpHR3.0-EYFP 23 (link) fragments, were each cloned into a Rosa26-pCAG-LSL-WPRE-bGHpA targeting vector 9 (link), in between LSL and WPRE sequences. LSL sequence contains specifically LoxP – Stop codons – 3x SV40 polyA – LoxP.
The targeting vectors were linearized and transfected into the 129/B6 F1 hybrid ES cell line G4 44 (link). G418-resistant ES clones were first screened by PCR using primers spanning the 1.1 kb 5’ genomic arm (forward primer: 5’-gggctccggctcctcagaga-3’, reverse primer: 5’-atgccaggcgggccatttac-3’), and then confirmed by Southern blot analysis of HindIII digested DNA, which was probed with a 1.1 kb genomic fragment from immediately upstream of the 5’ arm. Positive ES clones were injected into C57BL/6J blastocysts to obtain chimeric mice following standard procedures. Chimeric mice were bred with C57BL/6J mice to obtain germline transmitted F1 mice. The reporter mice can be bred with the Rosa26-PhiC31 mice (JAX Stock # 007743) 45 (link) to delete the PGK-neo cassette in the germline of the mice.
The targeting vectors were linearized and transfected into the 129/B6 F1 hybrid ES cell line G4 44 (link). G418-resistant ES clones were first screened by PCR using primers spanning the 1.1 kb 5’ genomic arm (forward primer: 5’-gggctccggctcctcagaga-3’, reverse primer: 5’-atgccaggcgggccatttac-3’), and then confirmed by Southern blot analysis of HindIII digested DNA, which was probed with a 1.1 kb genomic fragment from immediately upstream of the 5’ arm. Positive ES clones were injected into C57BL/6J blastocysts to obtain chimeric mice following standard procedures. Chimeric mice were bred with C57BL/6J mice to obtain germline transmitted F1 mice. The reporter mice can be bred with the Rosa26-PhiC31 mice (JAX Stock # 007743) 45 (link) to delete the PGK-neo cassette in the germline of the mice.
