Lenti x provirus quantitation kit

Twelve mounted histological slides were immersed in xylene for 2 days and then hydrated with ethanol solutions and water. Lentiviral-injected striata were removed with a scalpel and DNA extraction was performed using the GeneRead DNA FFPE Kit (Qiagen), starting from step 6 of manufacturer’s protocol. Purified cellular genomic DNA was then quantified by optical density (OD) using a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and the purity was evaluated by measuring the ratio of OD at 260 and 280 nm. Copy number of integrated lentiviruses (proviruses) present in the transduced cells of striatum was detected by qPCR using the Lenti-X Provirus Quantitation Kit (Takara) and according to the manufacturer’s instructions. Briefly, serial dilutions of the cellular gDNA were subjected to qPCR amplification alongside dilutions of a calibrated Provirus control template. The final result was expressed in terms of provirus copies (vg)/cell.

RNA was isolated with TRIzol RNA Isolation Reagents (Thermo Fisher). Reverse transcription was performed with PrimeScript™ RT Reagent Kit (Takara), and quantitative PCR was performed with Power SYBR Green Master Mix (Thermo Fisher). For quantification, the 2^−ΔΔCt method was used to calculate the relative RNA levels against GAPDH. The provirus content in cells that have been transduced with lentivirus was detected using Lenti-X provirus quantitation kit (631239, Takara) according to the manufacturer’s instruction. The copy number of proviruses in cells was determined by detecting integrated proviruses in genomic DNA using real-time quantitative PCR. The sequences of qPCR primers are shown in Supplementary Table 1.

We have designed a LV vector containing the human FAH gene under control of a liver-specific promoter (HCR-AAT; hepatic control region enhancer and alpha-1 antitrypsin promoter), currently being trialed in humans for the treatment of hemophilia B^{40 (link)}. A schematic representation of this vector is provided in Fig. 1a. In order to generate viral vectors, the LV-SFFV-eGFP or LV-AAT-huFAH expression construct, together with the packaging plasmid p8.91 and the vesicular stomatitis virus glycoprotein G-encoding plasmid pVSV-G, was transfected into 293 T/17 cells (CRL-11268, ATCC, Manassas, VA) using 1 mg/ml polyethylenimine (Polysciences, Warrington, PA). Viral supernatant was harvested 48 and 72 h after transfection, filtered through a 0.45-μm filter, and concentrated by ultracentrifugation (70,000 g, 1.5 h at 4 °C). After resuspension in serum-free media (DMEM, Thermo Fisher Scientific, Waltham, MA), LV vectors were aliquoted and stored at −80 °C. Vector titers were determined by p24 enzyme-linked immunosorbent assay and qPCR using the Lenti-X Provirus Quantitation Kit (Clontech, Mountain View, CA).

An expression vector encoding the human codon-optimized ANDV GPC gene was synthesized (DNA2.0, Menlo Park, CA, USA), and HIV pseudoparticles bearing ANDV GPC were prepared as described previously [26 (link)]. Briefly, 1 × 10⁶ 293T-LentiX cells (Clontech, Mountain View, CA, USA) were co-transfected with plasmid DNA encoding the HIV genome containing the firefly luciferase gene (pNL4-3.Luc.R^-E^-) and an expression vector encoding the vesicular stomatitis virus (VSV) G protein or ANDV GPC. The ratio of HIV plasmid to glycoprotein expression vector was 8:1. Pseudoparticles were harvested from the medium of 293T-LentiX cells 48 and 72 h post transfection, and supernatants were combined and filtered to remove cellular debris. Pseudoparticle titers were determined on HMVEC-L cells using the LentiX provirus quantitation kit (Clontech). To measure ANDV glycoprotein-dependent entry, pseudoparticles were added to HMVEC-L cells and allowed to adsorb for 6 h. After this time, the inoculum was removed and replaced with phenol red-free EGM-2-MV medium (Lonza). Two days after infection, firefly luciferase levels were measured using the luciferase assay system (Promega, Madison, WI, USA).