Table 1 Viral combinations used in this study and their functions.
From: Cell- and region-specific miR30-based gene knock-down with temporal control in the rat brain
Abbreviation | Vector combination | Function |
|---|---|---|
LVVs-miRLuc-neurone | LV-SYN-Luc + LV-Tretight-GFP-miR30-shRNA/Luc + LV-mCMV/SYN-tTA | Neurone-specific Luc knock-down system. Tet-off transactivator tTA is expressed from LV-mCMV/SYN-tTA which contains the bidirectional amplified SYN promoter to ensure high level of tTA. tTA binds to Tretight promoter in LV-Tretight-GFP-miR30-shRNA/Luc and activates the expression of shRNA/Luc. |
LVVs-miRLuc-glia | LV-GfaABC1D-Luc +LV-Tretight-GFP-miR30-shRNA/Luc +LV-mCMV/GfaABC1D-tTA | Astrocyte-specific Luc knock-down system. Transactivator tTA is expressed from LV-mCMV/GfaABC1D-tTA which contains the bidirectional amplified GfaABC1 D promoter to ensure high level of tTA. tTA binds to Tretight promoter in LV-Tretight-GFP-miR30-shRNA/Luc and activates the expression of shRNA/Luc. |
LVVs-miRLuc-control1 | LV-SYN-Luc + LV-Tretight-GFP-miR30-shRNA/Luc + LV-SYN-WPRE | Control combination used in Luc knock-down experiments for the neurone-specific system. Since there is no tTA expression in this combination, shRNA/Luc will not be expressed. So this combination marked the background expression level of Luc driven by SYN promoter. |
LVVs-miRLuc-control2 | LV-GfaABC1D-Luc + LV-Tretight-GFP-miR30-shRNA/Luc + LV-GfaABC1D-WPRE | Control combination used in Luc knock-down experiments for the astrocyte-specific system. Similar as LVVs-miRLuc-control 1, there is no tTA expression in this combination; shRNA/Luc will not be expressed. Therefore this combination marked the background expression level of Luc driven by GfaABC1 D promoter. |
LVVs-miRnNOS-neurone | LV-Tretight-GFP-miR30-shRNA/nNOS + LV-mCMV/SYN-tTA | Neurone-specific nNOS knock-down system. Mechanism is similar as LVVs-miRLuc-neurone. |
LVVs-miRnNOS-glia | LV-Tretight-GFP-miR30-shRNA/nNOS + LV-mCMV/GfaABC1D-tTA | Astrocyte-specific Luc knock-down system. Mechanism is similar as LVVs-miRLuc-glia. |
LVVs-miRnNOS-control1 | LV-Tretight-GFP-miR30-shRNA/nNOS + LV-SYN-WPRE | Control combination used in nNOS knockdown experiments for the neurone-specific system. Mechanism is similar as LVVs-miRLuc-control1. |
LVVs-miRnNOS-control2 | LV-Tretight-GFP-miR30-shRNA/nNOS + LV-GfaABC1D-WPRE | Control combination used in nNOS knockdown experiments for the astrocyte-specific system. Mechanism is similar as LVVs-miRLuc-control2. |
LVVs-miRnNOS-negative control1 | LV-Tretight-GFP-miR30-shRNA/Luc + LV-mCMV/SYN-tTA | Negative control combination used in nNOS knock-down experiments for the neurone-specific system. Instead of producing shRNA/nNOS it produces shRNA/Luc which shouldn't work on nNOS knockdown. |
LVVs-miRnNOS-negative control2 | LV-Tretight-GFP-miR30-shRNA/Luc + LV-mCMV/GfaABC1D-tTA | Negative control combination used in nNOS knock-down experiments for the astrocyte-specific system. Instead of producing shRNA/nNOS it produces shRNA/Luc which shouldn't work on nNOS knockdown. |