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Generation of ROSA26lSlrSrZsGreen (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-ZsGreen) mice has been described in a previous study20. These mice have been crossed to a ubiquitously expressed Deleter-Cre line to obtain R26rSrZsGreen mice20. ROSA26rSrlSltdTomato (ROSA26-CAGS-rox-STOP-rox-lox-STOP-lox-tdTomato-WPRE) mice were purchased from The Jackson Laboratory.

The knock-in lines ROSA26lSlrSrZsGreen (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-ZsGreen), ROSA26lSlrSrhM3Dq (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-hM3Dq-2A-ZsGreen-WPRE) and ROSA26lSlrSrEGFPL10a (ROSA26-CAGS-lox-STOP-lox-rox-STOP-rox-EGFPL10a-WPRE) were generated in-house. For generation of these mouse lines, a ROSA26 locus-targeting vector (B9-36) was designed in which both a loxP-flanked STOP cassette and a rox-flanked STOP cassette prevent CAGS promoter-driven expression of the corresponding functional transgenic construct. For hM3Dq, the 5′-primer (5AscRassle) overhang used for the amplification of the transgene contained an AscI site and a Kozak consensus sequence and the 3′-primer (3AscRassle) overhang contained an AscI site and one C to stay in frame for the 2A-ZsGreen translation. For the EGFPL10a, the 5′-primer overhang used for the amplification of the transgene contained an AscI site and a Kozak consensus sequence and the 3′-primer overhang contained an XmaI. The sequence-verified knock-in sequences of hM3Dq and EGFPL10a were cloned into the AscI-digested and AscI/XmaI-digested B9-36-targeting constructs, respectively. After vector transfection into Bruce 4 embryonic stem cells, they were subsequently screened for correct integration by standard Southern blot methods. To this end, a ROSA26 probe was used45 on EcoRI-digested clonal genomic DNA that indicated homologous recombination via detection of an additional 7.1-kb band besides the 16-kb endogenous ROSA26 band. To exclude random integration, a Neo probe was used that detected one single 7.1-kb band in case of a single correct ROSA26 insertion. Correctly targeted and verified embryonic stem cell clones were chosen for blastocyst injection carried out by Taconic Biosciences to obtain chimeric animals. Resulting chimeras were backcrossed with C57BL/6N mice to obtain germline transmission on a pure C57BL/6N background.

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