Lentiviral Transduction of ERG Variants

For transfection-based reporter assays, human GR and MR were cloned into pACT (Promega) as N-terminal VP16 fusions using HiFi assembly (NEB). The human ERG ETS domain, and human VP16-AR WT and mutants were cloned into pCDNA3.1 using HiFi assembly. All other constructs were described previously (Wasmuth et al., 2020 (link)). For lentiviral transduction of ERG variants in mouse prostate organoids, modified derivatives of pLVX-TRE3G-IRES (Takara) were engineered for constitutive expression by replacing the TRE3G element with a UBC promoter. To construct LVX-eGFP-ERG-PuroR variants, eGFP was cloned into MCS I; human ERG variants were cloned via HiFi assembly into MCS II.

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Wasmuth E.V., Vanden Broeck A., LaClair J.R., Hoover E.A., Lawrence K.E., Paknejad N., Pappas K., Matthies D., Wang B., Feng W., Watson P.A., Zinder J.C., Karthaus W.R., de la Cruz M.J., Hite R.K., Manova-Todorova K., Yu Z., Weintraub S.T., Klinge S, & Sawyers C.L. (2022). Allosteric interactions prime androgen receptor dimerization and activation. Molecular cell, 82(11), 2021-2031.e5.

Publication 2022

HiFi assembly pLVX-TRE3G-IRES

Assays Derivatives Ets domain Human Ires Mouse Organoids Promega Prostate Transfection Vp16

Corresponding Organization : Howard Hughes Medical Institute

Other organizations : Janelia Research Campus, The University of Texas Health Science Center at San Antonio

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Variable analysis

independent variables

Human GR and MR cloned into pACT (Promega) as N-terminal VP16 fusions
Human ERG ETS domain cloned into pCDNA3.1
Human VP16-AR WT and mutants cloned into pCDNA3.1
ERG variants for lentiviral transduction in mouse prostate organoids

dependent variables

Outcomes measured in transfection-based reporter assays

control variables

All other constructs described previously (Wasmuth et al., 2020)

controls

Positive controls: Not specified
Negative controls: Not specified

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