Generation of Rosa26-based Optogenetic Mouse Line

In this study, Rosa26-CAGp-LSL-ACR2-eYFP (Gt(ROSA)26Sor^{tm1(CAG-ACR2/EYFP)Ksak}, or LSL-ACR2) was newly generated using a method similar to that used to generate the CAG-floxed STOP tdTomato reporter line (MGI: 6192640) in a previous study^{34 (link)}. Briefly, we constructed the targeting vector containing a CAG promoter^{35 (link)}, frt flanked pgk-Neo cassette^{36 (link)}, STOP cassette consisting of the terminator of the yeast His3 gene and SV40 poly-adenylation sequence^{37 (link)}, cDNA encoding ACR2 tagged with EYFP at the C-terminus from pFUGW-hGtACR2-EYFP (Addgene: Plasmid #67877), woodchuck hepatitis virus posttranscriptional regulatory element^{38 (link)}, and rabbit b-globin poly-adenylation sequence^{39 (link)}. Two loxP sites were inserted before the frt-Neo cassette and after the STOP cassette^{37 (link)}. This vector also exhibits 5’ and 3’ homology arms of 4.7- and 5.2-kb, respectively, which target the Xba1 site of intron 1 at the Rosa26 locus^{40 (link)}. The targeting vector (DDBJ: LC744045) was linearized and electroporated into the RENKA C57BL/6 embryonic stem cell line^{41 (link)}. G418-resistant ES clones were screened by Southern blot analysis for homologous recombination at the Rosa26 locus. Targeted ES clones were injected into eight-cell stage CD-1, which were cultured to produce blastocysts and later transferred to pseudopregnant CD-1 females. The resulting male chimeric mice were crossed with female C57BL/6 mice to establish the LSL-ACR2 line. The LSL-ACR2 mice used in the present study exhibit the Neo cassette. Previous studies showed that there is no difference in Rosa26 reporter expression with or without removal of the Neo cassette^{42 (link), 43 (link)}. Therefore, we did not test removal of the Neo cassette in the present study. The LSL-ACR2 mouse strain was raised in an inbred-manner for 6 to 13 generations after introduction into our animal facility. All progenies of LSL-ACR2 mice crossed with Cre-driver mice showed consistent expression of ACR2 (n = 20 animals). To express ACR2 in NA neurons, noradrenaline transporter (NAT)-Cre (Tg(Slc6a2-cre)FV319Gsat) mice^{14 (link)} and LSL-ACR2 mice were crossed to generate NAT-Cre;LSL-ACR2 (NAT-ACR2) mice, which were used for optogenetic experiments (total 14 animals) and immunohistochemistry (3 animals). To express tdTomato in NA neurons, Ai14 (B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J) mice^{42 (link)} and NAT-Cre mice were crossed to generate NAT-Cre;Ai14 (NAT-tdTomato) mice, which were used for negative control experiments in vivo (total 6 animals). To express ACR2 in Vglut2-positive glutamatergic, Vgat-positive GABAergic, or DAT-positive dopaminergic neurons, Vglut2-Cre (Slc17a6^tm2(cre)Lowl)^{44 (link)}, Vgat-Cre (Slc32a1^tm2(cre)Lowl)^{44 (link)}, or DAT-Cre (Slc6a3^{tm1.1(cre)Bkmn})^{45 (link)} mice were crossed with LSL-ACR2 mice to generate Vglut2-Cre;LSL-ACR2, Vgat-Cre;LSL-ACR2, or DAT-Cre;LSL-ACR2 mice, respectively, which were subsequently used to confirm expression (one animal for each strain). To confirm lack of ACR2 expression in Cre-negative animals, LSL-ACR2 mice (4 animals) and NAT-Cre mice (2 animals) were used. Adult mice (aged > 6 weeks) were used. Animals were housed at 23 ± 2 °C with a 12-h light–dark cycle, and feeding and drinking were available ad libitum. All experiments were carried out following the ARRIVE guidelines 2.0^{46 (link)} and the Nagoya University Regulations on Animal Care and Use in Research, and were approved by the Institutional Animal Care and Use Committees of the Research Institute of Environmental Medicine, Nagoya University, Japan (approval R210096 and R210729).