Table 1 Plasmids used and designed in this study

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unpublished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 P_ETRON8

[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: P_hCMV*-1-SEAP-IRES_PV-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unpublished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 P_ETR 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-P_ETR-IRES_PV-IRES_EMCV-pA_SV40-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-P_ETR-Intron_β-globin-ET1-pA_hgh-ITR).

this work

pDF54

AAV2 vector encoding EYFP driven by the erythromycin responsive P_ETR promoter. P_ETR was excised from pDF55 using AccI/NheI and cloned into the corresponding sites of pDF60, thus resulting in pDF54 (ITR-P_ETR-EYFP-pA_SV40-ITR).

this work

pDF55

AAV2 vector encoding divergent expression units for ECFP driven by P_ETR 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-pA_I-ECFP←P_ETR-ETR-P_HSP70min→ET1-pA_SV40-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-P_SV40-ET1-pA_SV40-ITR).

this work

pDF60

AAV2 vector encoding EYFP driven by a constitutive hCMV promoter. P_hCMV-EYFP was excised from pMF351 using XbaI/PacI and cloned into NheI/PacI sites of pDF63, thus resulting in pDF60 (ITR-P_hCMV-EYFP-pA_SV40-ITR).

this work

pDF61

AAV2 vector encoding SEAP driven by an erythromycin responsive P_ETR promoter. SEAP was excised from pCF019 using NheI/ClaI and cloned into the NheI/BstBI sites of pDF54, thus resulting in pDF61 (ITR-P_ETR-SEAP-pA_SV40-ITR).

this work

pDF63

AAV2 vector containing an SV40 promoter followed by an IRES_PV and a IRES_EMCV element. P_SV40-IRES_PV-IRES_EMCV 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- P_SV40-IRES_PV-IRES_EMCV-pA_SV40-ITR).

this work

pDF74

AAV2 vector containing tricistronic expression cassette driven by a P_ETRON8 promoter. The P_ETRON8 promoter was excised from pWW76 using NheI/EcoRI and cloned into the corresponding sites of pDF63, thus resulting in pDF74 (ITR-P_ETRON8-IRES_PV-IRES_EMCV-pA_SV40-ITR).

this work

pDF75

AAV2 vector encoding dicistronic expression unit consisting of SEAP followed by an IRES_PV element followed by ET1 driven by P_ETR. Dicistronic expression cassette was excised from pBP141 using XbaI/PacI and cloned into the NheI/PacI sites of pDF54, thus resulting in pDF75 (ITR-P_ETR-SEAP-IRES_PV-ET1-pA_SV40-ITR).

this work

pDF76

AAV2 vector encoding SEAP driven by a P_ETRON8 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-P_ETRON8-SEAP-IRES_EMCV-pA_SV40-ITR).

this work

pDF77

AAV2 vector encoding SEAP under the control of an erythromycin responsive P_ETR promoter (additional upstream ATG deleted). P_ETR was excised from pDF54 using AccI/EcoRI and cloned into the ClaI/EcoRI sites of pDF61, thus resulting in pDF77 (ITR-P_ETR-SEAP-pA_SV40-ITR)

this work

pDF89

AAV2 vector encoding divergent expression units for EYFP driven by P_ETR 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-pA_I-EYFP←P_ETR-ETR-P_HSP70min→ET1-pA_SV40-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-P_hCMV-Intron_β-globin-SEAP-pA_hgh-ITR)

pDF124

AAV2 vector encoding dicistronic expression unit consisting of EYFP followed by an IRES_EMCV 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-P_ETR-EYFP-IRES_EMCV-ET1-pA_SV40-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-P_hCMV-Intron_β-globin-E-KRAB-pA_hgh-ITR).

this work

pDF141

AAV2 vector encoding self-regulated expression cassette consisting of ET1 driven by a constitutive SV40 promoter and EYFP driven by P_ETR. 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-P_SV40-ET1-pA_SV40-P_ETR-EYFP-pA_SV40-ITR).

this work

pDF143

AAV2 vector encoding self-regulated expression cassette consisting of ET1 driven by a constitutive SV40 promoter and SEAP driven by P_ETR. The SEAP containing insert was excised from pDF77 using KpnI/SpeI and cloned into the corresponding sites of pDF141, thus resulting in pDF143 (ITR-P_SV40-ET1-pA_SV40-P_ETR-SEAP-pA_SV40-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-P_ETRON2-SEAP-IRES_EMCV-pA_SV40-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-P_ETRON4-SEAP-IRES_EMCV-pA_SV40-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-P_ETRON8-EYFP-pA_SV40-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-P_ETRON4-EYFP-pA_SV40-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-P_ETRON2-EYFP-pA_SV40-ITR).

this work