Cloning of AAV-based optoGenetic Vectors

To obtain pAAV-hSyn-GFP-IRES-tTA vector, DNA fragment of GFP-IRES-tTA from pAAV-TRE-fDIO-GFP-IRES-tTA^{66 (link)} was inserted into the pAAV-hSyn-DIO-hM3D(Gq)-mCherry^{67 (link)} lacking DIO-hM3D(Gq)-mCherry region by SalI and EcoRV digestion using the NEBuilder HiFi DNA assembly (NEB). DNA fragment of the hSyn promoter regions was exchanged for the L7 minimal promoter^{68 (link)} to construct pAAV-L7-GFP-IRES-tTA.
To obtain AAV-TRE-HA-mGlu1(WT) or AAV-TRE-HA-mGlu1(N264H), DNA fragment of TRE promoter and WPRE3^{35 (link)} were obtained from pAAV-TRE-fDIO-GFP-IRES-tTA^{66 (link)}, and SV40 late polyA was from pCI-neo vector (Promega). DNA fragment of HA-tagged mGlu1 was obtained by inserting HA-tag after the membrane localization signal of mGlu1. These DNA fragments were inserted into pAAV-hSyn-DIO-hM3D(Gq)-mCherry^{67 (link)} lacking DNA fragment from hSyn promoter to hGH polyA by MluI and PmlI digestion using the NEBuilder HiFi DNA assembly (NEB). See Supplementary Fig. 14a for illustration of these vector constructs. pAAV-hSyn-DIO-hM3D(Gq)-mCherry and pAAV-TRE-fDIO-GFP-IRES-tTA are gifts from Bryan Roth (Addgene plasmid #44361) and Minmin Luo (Addgene plasmid #118026), respectively.

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Ojima K., Kakegawa W., Yamasaki T., Miura Y., Itoh M., Michibata Y., Kubota R., Doura T., Miura E., Nonaka H., Mizuno S., Takahashi S., Yuzaki M., Hamachi I, & Kiyonaka S. (2022). Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue. Nature Communications, 13, 3167.

Publication 2022

NEBuilder HiFi DNA assembly pCI-neo vector

Digestion Gifts Ires Membrane Plasmid Polya Promega Sv40 Vector

Corresponding Organization : Nagoya University

Other organizations : Keio University, Kyoto University, University of Tsukuba

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Variable analysis

independent variables

Insertion of GFP-IRES-tTA DNA fragment into pAAV-hSyn-DIO-hM3D(Gq)-mCherry vector
Replacement of hSyn promoter regions with L7 minimal promoter to construct pAAV-L7-GFP-IRES-tTA
Insertion of TRE promoter, WPRE3, and SV40 late polyA into pAAV-hSyn-DIO-hM3D(Gq)-mCherry vector to obtain AAV-TRE-HA-mGlu1(WT) or AAV-TRE-HA-mGlu1(N264H)

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive controls: pAAV-hSyn-DIO-hM3D(Gq)-mCherry and pAAV-TRE-fDIO-GFP-IRES-tTA vectors (gifts from Bryan Roth and Minmin Luo, respectively)

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