Plvx tetone

SMN1 cDNA was amplified using Turbo Pfu (Stratagene) and inserted into a modified pLVX doxycycline-inducible vector containing a MYC-tagged BirA using pLVX-TetOne (631849; Clontech) and BirA. SMN1 was also inserted in pGEX-6P1 using BamHI and XhoI, and in pET28a containing an intein sequence and chitin-binding protein tag (MxE-CBP) using BamHI and NotI. cDNAs from candidate genes were cloned from the total RNA extracted from GM03813 cells (Coriell Institute) using TRIzol (Invitrogen) after reverse transcription using VILO (Invitrogen) and then inserted into pCMV 3×FLAG (Stratagene) using BamHI (New England Biolabs) and XhoI (Promega). All constructs were sequence-verified by Sanger sequencing services from Biofidal. The lentiviral packaging plasmids pMD2.G (12259; Addgene) and psPAX2 (12260; Addgene) were provided by Dr Didier Trono. Sequences for primer sets used to amplify cDNA of interest or site-directed mutagenesis can be provided upon request.

pLVX-TetOne-Puro (Clontech/Takara, 631,847) with puromycin selection was used as the backbone vector for an inducible gene expression system. Human PARG cDNA were cloned to pLVX-TetOne-Puro, followed by P2A cleavage site and mClover3 gene that encodes fluorescence protein with NLS signal for the visual control of the promoter activity.
Packaging Lenti-X Vectors into lentiviral particles and virus tittering: The lentiviral construct, pLVX-TetOne-hPARG-mClover3-NLS-Puro was packaged into lentiviral particles using Lenti-X Packaging Single Shots Protocol from Clontech/Takara. Lentiviral vector stocks were collected and concentrated using the Lenti-X Concentrator Protocol (Clontech/Takara). Lenti-X virus stocks were tittered using puromycin selection with HT1080 cells; the titer of virus corresponded to the number of colonies generated by the highest dilution multiplied by the dilution factor.
ccRCC cell lines were infected with lentivirus, selected with puromycin and single clones were generated using FACS sorting or with cloning rings. Clones with the highest fold change of hPARG induction vs. control detected with Western blotting were selected for propagation and further experiments.

Mouse Glis2 (NCBI Accession NM_031184) was cloned into several vectors with different epitope tag combinations: pLenti CMV GFP Blast (Addgene, Cat. no. #17445) with N-terminal V5 tag and C-terminal EGFP tag; pLVX-TetOne (Takara, Cat. no. 631846) with C-term FLAG tag; pHTC HaloTag® CMV-neo was used to clone Glis2 with C-term Halo tag. Human GLIS2 (NCBI Accession NM_032575) was purchased as GLIS2-HaloTag® human ORF in pFN21A (Promega, Cat. no. FHC03277). Mouse Glis3 (NCBI Accession NM_175459) was cloned into pLenti CMV GFP Blast with C-term EGFP tag. The cilia marker Nphp3^(1-200)-mApple was made by amplifying a fragment corresponding to amino acid 1-200 of mouse Nphp3 (NCBI Accession NM_028721.3) from mouse kidney cDNA and combining it in-frame with mApple using PCR, followed by subcloning into a pCDH-Hygro vector which was modified from pCDH-EF1-MCS-IRES-Puro (System Biosciences). All constructs were validated by sequencing and immunoblot expression analysis. Transient transfection of HEK293T cells was done with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. no. 11668030). Stable gene expression was achieved using lentivirus transduction and selection with appropriate antibiotic.