HEK293T cells were transiently cotransfected with lentiviral packaging plasmids pVSV-G, pMDLg, p-RSV-Rev, and pLKO.1 vectors (Sigma-Aldrich) containing a specific shRNA sequence directed against human Skap2 (5′-CCGGGCAGATGTTGAAACATTTGTA CTCGAGTACAAATGTTTCAACATCTGCTTTTT-3′), human WASp (5′-CCGGCGAGACCT CTAAACTTATCTACTCGAGTAGATAAGTTTAGAGGTCTCGTTTTT-3′), or control (5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′). Infection of promyelocytic HL60 cells was performed by incubation with medium containing lentiviral particles for 48 h. Stable knockdown of Skap2 and WASp in transduced HL60 cells was confirmed by Western blotting and maintained by puromycin selection (1 µg/ml; Sigma-Aldrich).
P rsv rev
Manufactured by Merck Group
P-RSV-Rev is a laboratory equipment product. It is designed to perform a specific core function, which is to detect and analyze Respiratory Syncytial Virus (RSV) in samples. The product is intended for use in research and diagnostic settings, but no further details on its intended use or application can be provided.
Automatically generated - may contain errors
Lab products found in correlation
Plko 1, by Merck Group (1 mentions)
Puromycin selection, by Merck Group (1 mentions)
Plenti puro vector, by Addgene (1 mentions)
Polybrene, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pmd2 g, by Merck Group (1 mentions)
Plenti puro, by Merck Group (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Plenti puro, by Addgene (1 mentions)
Xbai, by Takara Bio (1 mentions)
Pmdlg prre, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Prsv rev, by Addgene (1 mentions)
2 protocols using p rsv rev
1
Lentiviral Knockdown of Skap2 and WASp in HL60 Cells
2
Lentiviral Plasmid-Based Gene Transduction
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All third-generation lentiviral plasmids were obtained from Addgene (Watertown, MA, USA). The envelope (pMD2.G; Addgene plasmid #12259; http://n2t.net/addgene:12259; RRID: Addgene_12259) and packaging plasmids (pRSV-Rev; Addgene plasmid #12253; http://n2t.net/addgene:12253; RRID: Addgene_12253 and pMDLg/pRRE; Addgene plasmid # 12251, http://n2t.net/addgene:12251; RRID: Addgene_12251) were gifts from Dr Tronoa (32 (link)). The transfer plasmid was a gift from Ie-Ming Shih (33 (link)) (pLenti-puro; Addgene plasmid #39481; http://n2t.net/addgene:39481; RRID: Addgene_39481). The pLenti-puro FLAG-tagged FOXL2 was generated by PCR using pcDNA3 FLAG-tagged FOXL2 as the template and the primers FLAG-FOXL2-F and FLAG-FOXL2-R. The PCR products were digested with BamHI and XbaI (Takara Bio) and ligated into the pLenti-puro vector (Addgene). 293T cells were used for lentivirus production by transfection of 12 μg of pMDLg/pRRE and pRSV-Rev, 8 μg of pMD2.G and 16 μg of pLenti-puro empty vector or pLenti-puro-FLAG-FOXL2 for 48 h. Viral supernatant was collected for KGN cell infection in the presence of 10 μg/ml polybrene (H9268, Sigma-Aldrich) (0.5 ml of lentivirus to 5 × 104 cells in 1.5 ml of medium). After 48 h of infection, cells were selected with 2 μg/ml puromycin (P8833, Sigma-Aldrich) for 30 days.
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