Protocol detail

Lentiviral shRNA and Overexpression Knockdown

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Lentiviral clones expressing two, non-overlapping shRNAs directed against human c-Myc (TRCN0000039640, TRCN0000039642), human HMGCR (TRCN0000233114, TRCN0000233115) or a non-targeting control shRNA that has no targets in human genomes (shCONT: SHC002) were obtained from Sigma-Aldrich (St. Louis, MO). shRNAs with non-overlapping sequences that had the best relative knockdown efficiency were used for all experiments. A lentiviral overexpression plasmid for MYC was generated by cloning the ORF with the N-terminal Flag into the pCDH-MCS-T2A-Puro-MSCV vector (System Biosciences) (24 (link)). Lentiviral particles were generated in 293FT cells in Neurobasal complete medium (Life Technologies) with co-transfection of the packaging vectors pCMV-dR8.2 dvpr and pCI-VSVG (Addgene) using a standard calcium phosphate transfection method.

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Wang X., Huang Z., Wu Q., Prager B.C., Mack S.C., Yang K., Kim L.J., Gimple R.C., Shi Y., Lai S., Xie Q., Miller T.E., Hubert C.G., Song A., Dong Z., Zhou W., Fang X., Zhu Z., Mahadev V., Bao S, & Rich J.N. (2017). MYC-regulated Mevalonate Metabolism Maintains Brain Tumor Initiating Cells. Cancer research, 77(18), 4947-4960.

Publication 2017

shCONT: SHC002 pCDH-MCS-T2A-Puro-MSCV vector Neurobasal complete medium pCMV-dR8.2 dvpr pCI-VSVG

Calcium phosphate Cells Hmgcr Human Human genomes Plasmid Shrnas Transfection Transfection vectors Vector

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Variable analysis

independent variables

ShRNAs directed against human c-Myc (TRCN0000039640, TRCN0000039642)
ShRNAs directed against human HMGCR (TRCN0000233114, TRCN0000233115)
Non-targeting control shRNA (shCONT: SHC002)
MYC overexpression plasmid

dependent variables

Measured outcomes (not explicitly mentioned)

control variables

Neurobasal complete medium (Life Technologies)
Packaging vectors pCMV-dR8.2 dvpr and pCI-VSVG (Addgene)

controls

Positive control: Non-targeting control shRNA (shCONT: SHC002)
Negative control: Not explicitly mentioned

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