The coding cDNA sequence of MCC cloned from the human MM cell line LP1 cells was subcloned into the lentiviral expression vector pUB-eGFP-Thy1.1 [87 (link)] (generously provided by Dr. Zhibin Chen, the University of Miami, Miami, FL) by replacing the eGFP coding sequence with the MCC coding sequence. To facilitate immunoprecipitation experiments, we engineered an N-terminal FLAG tag or a C-terminal SBP-6×His tag [88 (link)] in frame with the MCC coding sequence, respectively. We subsequently generated two lentiviral expression vectors of tagged hMCC, including pUB-FLAG-hMCC and pUB-hMCC-SBP-6×His. Lentiviral shRNA vectors specific for human MCC (including hMCC shRNA 1332, 1388, 2284 and 2689; all in Torc1 vectors) or a scrambled shRNA vector were purchased from Sigma. To facilitate FACS analysis and cell sorting, we engineered an eGFP-expressing version of all the shRNA vectors by replacing the puromycin resistance gene of Torc1 with the eGFP coding sequence. Each lentiviral expression or shRNA vector was verified by DNA sequencing.
Lentiviral shrna vectors
Manufactured by Merck Group
Sourced in United States
Lentiviral shRNA vectors are a type of genetic engineering tool used for gene expression manipulation in cells. They are designed to deliver short hairpin RNA (shRNA) sequences into target cells, which can then be used to knockdown or silence the expression of specific genes. These vectors are based on the lentivirus, a type of retrovirus, which allows for stable and efficient transduction of both dividing and non-dividing cells.
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Lab products found in correlation
Pmd2 g, by Addgene (3 mentions)
Importin α1, by Merck Group (2 mentions)
Pmdlg prre, by Addgene (2 mentions)
Prsv rev, by Addgene (2 mentions)
Enhanced chemiluminescence substrate, by GE Healthcare (1 mentions)
Shc002, by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
5 protocols using lentiviral shrna vectors
1
Lentiviral Vectors for MCC Expression and Knockdown
2
Plasmid Construction for AMPK Studies
3
Plasmid Construction for AMPK Studies
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An expression plasmid for CA-AMPK53 (link) was kindly provided by Dr. Rusty Jones (McGill University) and the insert was transferred to pBabe-puro and pWZL-blast. Expression plasmids for mouse C/EBPβ coding region (C/EBPβΔUTR) and C/EBPβ coding region plus 3′UTR (C/EBPβUTR) have been described previously10 (link); the genes were transferred to pBabe-puro and pWZL-blast retroviral vectors. Lentiviral expression vectors for AMPKα2 and CaMKKβ were provided by Dr. Dominic Esposito (FNLCR, Frederick, MD). Lentiviral vectors containing an shRNA construct simultaneously targeting AMPKα1/2 (human and mouse)54 (link) and a control shRNA vector were kindly provided by Dr. Thomas Brown (Wright State University). Lentiviral shRNA vectors targeting mouse CaMKKβ (#TRCN0000276649), AMPKα1 (#TRCN0000360770) and Importin α1 (#TRCN0000093514) were purchased from Sigma-Aldrich. Human C/EBPβ shRNA and a non-targeting shRNA control in pSuperRetro-neo were described previously10 (link). Human HRASG12V was expressed from pWZL-hygro6 (link). Plasmids for retroviral packaging have been described9 (link); lentiviral packaging/envelope plasmids pMD2.G (#12259), pMDLg/pRRE (#12251), and pRSV/Rev (#12253) were obtained from Addgene.
4
Lentiviral Knockdown of NONO and XLF
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Gene silencing was performed using lentiviral shRNA vectors (Sigma, St. Louis, MO, USA). Two shRNAs were tested for each gene and the more effective was used (for NONO, NM_007363, TRCN0000286693
(dCCGGGCCAGAATTCTACCCTGGAAACTCGAGTTTCCAGGGTAGAATTCTGGCTTTTTG); for XLF, NM_024782, TRCN0000275632
(dCCGGTACCATGGACTTTAGGTATATCTCGAGATATACCTAAAGTCCATGGTATTTTT)). The control shRNA was SHC002 (Sigma). Lentiviruses were generated by Lipofectamine 3000-mediated triple co-transfection of human HEK293FT cells with shRNA plasmid, psPAX2 (Addgene # 12260), and pMD2.G (Addgene #12259). Media was changed at 18 h, cells were incubated for an additional 30 h, and virus-containing supernatant was harvested and centrifuged to remove debris (1000g at 4°C for 15 min). Attenuation of protein expression was confirmed by immunoblotting using rabbit anti-NONO (GeneTex, GTX63618), rabbit anti-XLF (Bethyl Laboratories, A300-730A), rabbit anti-β-actin (Sigma, A5060), and horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (GE Healthcare, NA 934V). Membranes were developed using Enhanced Chemiluminescence substrate (GE Healthcare) and were visualized using X-ray film.
(dCCGGGCCAGAATTCTACCCTGGAAACTCGAGTTTCCAGGGTAGAATTCTGGCTTTTTG); for XLF, NM_024782, TRCN0000275632
(dCCGGTACCATGGACTTTAGGTATATCTCGAGATATACCTAAAGTCCATGGTATTTTT)). The control shRNA was SHC002 (Sigma). Lentiviruses were generated by Lipofectamine 3000-mediated triple co-transfection of human HEK293FT cells with shRNA plasmid, psPAX2 (Addgene # 12260), and pMD2.G (Addgene #12259). Media was changed at 18 h, cells were incubated for an additional 30 h, and virus-containing supernatant was harvested and centrifuged to remove debris (1000g at 4°C for 15 min). Attenuation of protein expression was confirmed by immunoblotting using rabbit anti-NONO (GeneTex, GTX63618), rabbit anti-XLF (Bethyl Laboratories, A300-730A), rabbit anti-β-actin (Sigma, A5060), and horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (GE Healthcare, NA 934V). Membranes were developed using Enhanced Chemiluminescence substrate (GE Healthcare) and were visualized using X-ray film.
5
Characterization of Aggressive Breast Cancer Cell Lines
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Cell Lines, Cell Culture, and DNA Constructs 67NR, 66cl4, and 4T1 cell lines were cultured as previously described (Rose et al., 2007) . The 4T1-derived liver-aggressive (2776 and 2792), lung-aggressive (533 and 537), and bone-aggressive cell populations (592 and 593) were described previously (Tabarie `s et al., 2015) . Proliferation rates were determined by seeding cells in 6-well plates and performing cell counts every day over a 4-day period to determine population doublings. ROS levels were measured using MitoSOX Red mitochondrial superoxide indicator (Invitrogen, #M36008) following a 15 min incubation period, and Mean Fluorescence Intensity (MFI) determined by flow cytometry. HIF-1a knockdown was achieved using lentiviral shRNA vectors from the TRC shRNA collection (Sigma-Aldrich, St. Louis, MO; TRCN0000232220 [hp#1] and TRCN0000232222 [hp#2]). Lentiviral supernatants were generated as described (Huang et al., 2012) . PDK1 knockdown was achieved using the miR-30-adapted LMP retroviral vector system (Dickins et al., 2005) .
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