The doxycycline-inducible pLVX-TRE3G-ZsGreen1 (Takara Bio USA) vectors containing ZsGreen1-IRES-CYP3A4 and ZsGreen1-IRES-CYP3A5 are previously described.30 (link) All cloning primers are presented in Table S5 . For this study, the ZsGreen1-IRES-CYP3A cassettes were subcloned into pcDNA3 (Thermo Fisher Scientific, Waltham, MA) to achieve constitutive gene expression, with ZsGreen1 serving as a transfection reporter. ZsGreen1-IRES-CYP3A4 was amplified with primers oAH522 and oAH523, and ZsGreen1-IRES-CYP3A5 was amplified with primers oAH522 and oAH524. The amplicons were cloned into HindIII-digested pcDNA3 with NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Mutants were made with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) with the primers listed in Table S5 .
Protocol detail
Constitutive Expression of CYP3A Isoforms
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pLVX-TRE3G-ZsGreen1
pcDNA3
NEBuilder HiFi DNA Assembly Master Mix
QuikChange II Site-Directed Mutagenesis Kit
Cyp3a4
Cyp3a5
Doxycycline
Gene expression
Ires
Primers
Site directed mutagenesis
Transfection
Vectors
Corresponding Organization : St. Jude Children's Research Hospital
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Variable analysis
independent variables
- Doxycycline-inducible expression of ZsGreen1-IRES-CYP3A4 and ZsGreen1-IRES-CYP3A5 constructs in pcDNA3 vector
- Constitutive gene expression of CYP3A4 and CYP3A5
- ZsGreen1 expression as a transfection reporter
- PcDNA3 vector backbone
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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