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Table 1 Plasmids used and designed in this study

From: Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

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pSS134 Plasmid containing SEAP cassette unpublished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pBP141 Vector expressing SEAP and ET1 under tetracycline-responsive promoter: PhCMV*-1-SEAP-IRESPV-ET-pA unpublished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
  1. 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); IRES EMCV , internal ribosome entry site of encephalomyocarditisviral origin (502 bp); IRES PV , internal ribosome entry site of polioviral origin (635 bp); ITR, inverted terminal repeat (141 bp); KRAB, kruppel-associated box (450 bp); pA I , artificial polyadenylation signal (91 bp); pA hgh , human growth hormone polyadenylation signal (478 bp); pA SV40 , simian virus 40 polyadenylation signal (145 bp); P hEF1α , human elongation factor 1α promoter (1185 bp); P ETR , erythromycin responsive promoter (200 bp); P ETR ON, macrolide inducible promoter (530 bp); P hCMV , human immediate early cytomegalovirus promoter (663 bp); P HSP70min , heat shock protein 70 minimal promoter (350 bp); P SV40 , simian virus 40 promoter (308 bp); SEAP, human placental secreted alkaline phosphatase (1560 bp)
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BMC Biotechnology

ISSN: 1472-6750

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