Table 1.
Plasmids used and designed in this study
| Plasmid | Description and Cloning Strategy | Reference or source |
| pAAV-MCS | AAV transfer vector | Stratagene |
| pAAV-lacZ | AAV vector expressing lacZ | Stratagene |
| pDG | Helper construct encoding AAV Rep/Cap as well as Adeno virus E2A, E4 and VA. | [38] |
| pCF18 | Plasmid containing ECFP driven by a tetracycline responsive promoter and EYFP driven by a pristinamycin responsive promoter | [53] |
| pCF19 | Plasmid containing SEAP cassette | [54] |
| pCF125 | Plasmid expressing ECFP and ET1 from a bidirectional promoter | [21] |
| pSS134 | Plasmid containing SEAP cassette | unpublished |
| pWW43 | Plasmid expressing E-KRAB | [14] |
| pWW76 | Plasmid containing tricistronic expression configuration driven by PETRON8 | [14] |
| pWW78 | pTRIDENT1-based tricistronic expression vector for macrolide-responsive auto-regulated expression of up to two desired transgenes. | [55] |
| pBP141 | Vector expressing SEAP and ET1 under tetracycline-responsive promoter: PhCMV*-1-SEAP-IRESPV-ET-pA | unpublished |
| pMF123 | Plasmid encoding tricistronic expression cassette driven by a constitutive SV40 promoter. | [56] |
| pMF351 | Lentiviral vector encoding EYFP driven by a constitutive hCMV promoter | [51] |
| pDF37 | AAV2 vector containing a tricistronic PETR driven expression unit. The entire expression unit from pWW73 was excised using SspI/XbaI, polished by Klenow and cloned into pAAV-lacZ which was NotI digested and Klenow polished before, thus resulting in pDF37 (ITR-PETR-IRESPV-IRESEMCV-pASV40-ITR). | this work |
| pDF51 | AAV2 vector containing a constitutive hCMV driven ET1 cassette. ET1 was excised from pWW078 using EcoRI/HindIII and cloned into the corresponding sites of pAAV-MCS, thus resulting in pDF51 (ITR-PETR-Intronβ-globin-ET1-pAhgh-ITR). | this work |
| pDF54 | AAV2 vector encoding EYFP driven by the erythromycin responsive PETR promoter. PETR was excised from pDF55 using AccI/NheI and cloned into the corresponding sites of pDF60, thus resulting in pDF54 (ITR-PETR-EYFP-pASV40-ITR). | this work |
| pDF55 | AAV2 vector encoding divergent expression units for ECFP driven by PETR and ET1 driven by a HSP70 minimal promoter. The entire expression cassette was excised from pCF125 using EcoRV/XbaI and cloned into the HincII/SpeI sites of pDF60, thus resulting in pDF55 (ITR-pAI-ECFP←PETR-ETR-PHSP70min→ET1-pASV40-ITR). | |
| pDF56 | AAV2 vector encoding ET1 driven by a constitutive SV40 promoter. ET1 was excised from pWW78 using EcoRI/XbaI and cloned into the EcoRI/SpeI sites of pDF63, thus resulting in pDF56 (ITR-PSV40-ET1-pASV40-ITR). | this work |
| pDF60 | AAV2 vector encoding EYFP driven by a constitutive hCMV promoter. PhCMV-EYFP was excised from pMF351 using XbaI/PacI and cloned into NheI/PacI sites of pDF63, thus resulting in pDF60 (ITR-PhCMV-EYFP-pASV40-ITR). | this work |
| pDF61 | AAV2 vector encoding SEAP driven by an erythromycin responsive PETR promoter. SEAP was excised from pCF019 using NheI/ClaI and cloned into the NheI/BstBI sites of pDF54, thus resulting in pDF61 (ITR-PETR-SEAP-pASV40-ITR). | this work |
| pDF63 | AAV2 vector containing an SV40 promoter followed by an IRESPV and a IRESEMCV element. PSV40-IRESPV-IRESEMCV was excised from pMF123 using SspI/BglII, polished with Klenow and cloned into the polished NcoI/SpeI sites of pAAV-lacZ, thus resulting in pDF63 (ITR- PSV40-IRESPV-IRESEMCV-pASV40-ITR). | this work |
| pDF74 | AAV2 vector containing tricistronic expression cassette driven by a PETRON8 promoter. The PETRON8 promoter was excised from pWW76 using NheI/EcoRI and cloned into the corresponding sites of pDF63, thus resulting in pDF74 (ITR-PETRON8-IRESPV-IRESEMCV-pASV40-ITR). | this work |
| pDF75 | AAV2 vector encoding dicistronic expression unit consisting of SEAP followed by an IRESPV element followed by ET1 driven by PETR. Dicistronic expression cassette was excised from pBP141 using XbaI/PacI and cloned into the NheI/PacI sites of pDF54, thus resulting in pDF75 (ITR-PETR-SEAP-IRESPV-ET1-pASV40-ITR). | this work |
| pDF76 | AAV2 vector encoding SEAP driven by a PETRON8 promoter. SEAP was excised from pSS134 using EcoRI/HindIII and cloned into pDF37. This vector was digested using EcoRI/AscI and the SEAP containing insert was cloned into the corresponding sites of pDF74, thus resulting in pDF76 (ITR-PETRON8-SEAP-IRESEMCV-pASV40-ITR). | this work |
| pDF77 | AAV2 vector encoding SEAP under the control of an erythromycin responsive PETR promoter (additional upstream ATG deleted). PETR was excised from pDF54 using AccI/EcoRI and cloned into the ClaI/EcoRI sites of pDF61, thus resulting in pDF77 (ITR-PETR-SEAP-pASV40-ITR) | this work |
| pDF89 | AAV2 vector encoding divergent expression units for EYFP driven by PETR and ET1 driven by a HSP70 minimal promoter. The EYFP cassette was excised from pCF18 using AccI/EcoRV and cloned into the NruI/ClaI sites of pDF55, thus resulting in pDF89 (ITR-pAI-EYFP←PETR-ETR-PHSP70min→ET1-pASV40-ITR) | this work |
| pDF98 | Plasmid containing hEF1α promoter flanked by multiple cloning sites | unpublished |
| pDF109 | AAV2 vector encoding SEAP driven by a constitutive hCMV promoter. SEAP was excised from pDF61 using EcoRI/SpeI and cloned into the EcoRI/XbaI sites of pAAV-MCS, thus resulting in pDF109 (ITR-PhCMV-Intronβ-globin-SEAP-pAhgh-ITR) | |
| pDF124 | AAV2 vector encoding dicistronic expression unit consisting of EYFP followed by an IRESEMCV element followed by ET1. The IRES-ET1 containing insert was excised from pDF75 using HindIII, polished with Pfu polymerase, digested using BstXI and cloned into the SwaI/BstXI sites of pDF54, thus resulting in pDF124 (ITR-PETR-EYFP-IRESEMCV-ET1-pASV40-ITR). | this work |
| pDF126 | AAV2 vector encoding E-KRAB under the control of a constitutive hCMV promoter. The E-KRAB containing insert was excised from pWW043 using EcoRI/HpaI and cloned into the EcoRI/HincII sites of pAAV-MCS, thus resulting in pDF126 (ITR-PhCMV-Intronβ-globin-E-KRAB-pAhgh-ITR). | this work |
| pDF141 | AAV2 vector encoding self-regulated expression cassette consisting of ET1 driven by a constitutive SV40 promoter and EYFP driven by PETR. The entire ET1 expression cassette of pDF56 was excised using ClaI/PmlI and cloned into pDF54 which was digested by HindIII and polished by Pfu before, thus resulting in pDF141 (ITR-PSV40-ET1-pASV40-PETR-EYFP-pASV40-ITR). | this work |
| pDF143 | AAV2 vector encoding self-regulated expression cassette consisting of ET1 driven by a constitutive SV40 promoter and SEAP driven by PETR. The SEAP containing insert was excised from pDF77 using KpnI/SpeI and cloned into the corresponding sites of pDF141, thus resulting in pDF143 (ITR-PSV40-ET1-pASV40-PETR-SEAP-pASV40-ITR). | this work |
| pDF199 | AAV2 vector encoding SEAP under the control of a constitutive SV40 promoter followed by 2 binding sites for the transrepressor E-KRAB. The 4*ETR binding site containing fragment was excised from pWW55 using BstBI/NdeI and cloned into the corresponding sites of pDF76. Two of the binding sites were deleted by recombination during the cloning procedure, thus resulting in pDF199 (ITR-PETRON2-SEAP-IRESEMCV-pASV40-ITR). | this work |
| pDF200 | AAV2 vector encoding SEAP under the control of a constitutive SV40 promoter followed by 4 binding sites for the transrepressor E-KRAB. The 4*ETR binding site-containing fragment was excised from pWW55 using BstBI/NdeI and cloned into the corresponding sites of pDF76, thus resulting in pDF200 (ITR-PETRON4-SEAP-IRESEMCV-pASV40-ITR). | this work |
| pDF207 | AAV2 vector encoding EYFP under the control of a constitutive SV40 promoter followed by 8 binding sites for the transrepressor E-KRAB. EYFP was excised from pDF34 using EcoRI/PacI and cloned into the corresponding sites of pDF76, thus resulting in pDF207 (ITR-PETRON8-EYFP-pASV40-ITR). | this work |
| pDF208 | AAV2 vector encoding EYFP under the control of a constitutive SV40 promoter followed by 4 binding sites for the transrepressor E-KRAB. EYFP was excised from pDF34 using EcoRI/PacI and cloned into the corresponding sites of pDF200, thus resulting in pDF208 (ITR-PETRON4-EYFP-pASV40-ITR). | this work |
| pDF209 | AAV2 vector encoding EYFP under the control of a constitutive SV40 promoter followed by 2 binding sites for the transrepressor E-KRAB. EYFP was excised from pDF34 using EcoRI/PacI and cloned into the corresponding sites of pDF199, thus resulting in pDF209 (ITR-PETRON2-EYFP-pASV40-ITR). | this work |
Abbreviations: AAV2, adeno-associated virus type 2; ECFP, enhanced cyan fluorescent protein (720 bp); EM, erythromycin; ET1, erythromycin transactivator (972 bp); EYFP, enhanced yellow fluorescent protein (720 bp); IRESEMCV, internal ribosome entry site of encephalomyocarditisviral origin (502 bp); IRESPV, internal ribosome entry site of polioviral origin (635 bp); ITR, inverted terminal repeat (141 bp); KRAB, kruppel-associated box (450 bp); pAI, artificial polyadenylation signal (91 bp); pAhgh, human growth hormone polyadenylation signal (478 bp); pASV40, simian virus 40 polyadenylation signal (145 bp); PhEF1α, human elongation factor 1α promoter (1185 bp); PETR, erythromycin responsive promoter (200 bp); PETRON, macrolide inducible promoter (530 bp); PhCMV, human immediate early cytomegalovirus promoter (663 bp); PHSP70min, heat shock protein 70 minimal promoter (350 bp); PSV40, simian virus 40 promoter (308 bp); SEAP, human placental secreted alkaline phosphatase (1560 bp)