Peg ittm virus precipitation solution kit

Lentiviruses were produced by transient transfection of human embryonic kidney (HEK) 293FT cells with individual lentiviral vectors containing the pTRIPZ Doxycycline (Dox)-inducible transgene with packaging plasmids pMD2G and psPAX2 (Addgene, Cambridge, MA, USA) using Lipofectamine 2000 (Promega, Madison, WI, USA). Supernatants containing viral particles were collected 48 h post-transfection, filtered through 0.45 μm filters and viral particles concentrated using the PEG-itTM Virus Precipitation Solution Kit (System Biosciences, Mountain View, CA, USA). Cloning of the shRNA cassette sequences for eIF4G2/DAP5 (5′-TACCTCTAGTAATGGGCTTTA-3′) and non-silencing sequence control Nsi (5′-AATTCTCCGAACGTGTCACGT-3′) were previously described.^{18 (link)} Cells were infected with viral particles, selected with puromycin (1 μg/mL), and transformed with reporter plasmid expressing tRFP (Turbo Red Fluorescent Protein). The lentiviral vectors used for shNsi and shDAP5 contains a constitutively expressed transcript encoding the puromycin resistance gene, and a doxycycline-inducible cassette that expresses target shRNA sequence as well as the reporter protein tRFP (Turbo Red Fluorescent Protein). Cells were treated with 0.1–2 μg/mL of Dox and FACs isolated gating on RFP⁺. Silencing of DAP5 expression was confirmed by qRT-PCR and immunoblot analysis.