Clontech lenti x qrt pcr titration kit

Lentiviral vectors carrying the human TNF and IFNγ genes were produced as described elsewhere [21 (link)]. Briefly, 293T cells were transiently transfected with the HIV-1 vector plasmid pRRLsincppt-CMV-TNF-WPRE or pRRLsincppt-CMV-IFNƴ -WPRE), the packing vector containing HIV-1 gag/pol gene (pMD2-LgRRE), HIV-1 Rev (pRSV-Rev) and VSVG envelop plasmids using calcium phosphate. After 16 h the medium was replaced with fresh medium supplemented with 10 mM sodium butyrate. At 36 h, vector-containing medium was harvested and filtered through a 0.45-μm filter. The supernatant was then centrifuged overnight. The pellet was concentrated by ultracentrifugation and resuspended with TSSM (10 mM Tromethamine, 100 mM NaCl, 10 mg/ml sucrose and 10 mg/ml mannitol) over several hours. The genome copy number was calculated using the Clontech Lenti-X qRT-PCR Titration kit (Takara).

Lentiviral vectors carrying human TNFα and IFNγ were produced as described previously [24 (link)]. In summary, HEK-293 cells were transfected with HIV-1 transfer plasmid (pRRL-sincppt-CMV-TNF-WPRE or pRRL-sincppt-CMV-IFN-WPRE genome plasmid), the packaging vector plasmids expressing HIV-1 gag/pol gene (pMD2-LgRRE), VSV-G envelope plasmids (pMD2-VSV-G) and HIV-1 Rev (pRSV-Rev) using CaCl₂. Fresh medium supplemented with 10 mM sodium butyrate was added after 16 h. The supernatant was harvested and filtered through a 0.45 μm filter after 36 h, and centrifuged overnight. Lentiviral vectors were concentrated using ultracentrifugation and resuspended with TSSM (10 mM Tromethamine, 100 mM NaCl, 10 mg/mL sucrose and 10 mg/mL mannitol). The genome copy number was calculated using the Clontech Lenti-X qRT-PCR Titration kit (Takara Bio).

Lentiviral (LV) vectors expressing the human LTα (LVLTα), human IFNγ (LVIFNγ), or enhanced green fluorescent protein (LVGFP) genes were produced exactly as described previously [38 (link)], using the human immunodeficiency virus type 1 (HIV-1) transfer vector (pRRL-sincppt-CMV-eGFP-WPRE genome plasmid) with a human cytomegalovirus promoter (CMV) promotor. Complementary DNA sequences (cDNA) for human LTα or IFNγ were codon optimised for rat, including a 5′ Kozak sequence, and synthesised by Gene Art (Life Sciences, Paisley, UK). The biological and physical titres of the purified and concentrated vectors were calculated as described previously [38 (link)]. The LV genome copy number was calculated using the Clontech Lenti-X qRT-PCR Titration kit (Takara, Saint-Germain-en-Laye, France).