Lenti xtm qrt pcr titration kit

The lentiviral vectors were produced in HEK 293T cells as described previously^{45 (link)}. HEK 293 T cells were transfected with 15 μg lentiviral backbone vector and helper plasmids (pCAG-kGP1 10 μg, pCAG4-RTR2 5 μg, and pCAG-VSVG 5 μg) using a calcium phosphate transfection method. Ten to 16 hrs after transfection, culture medium was replaced with fresh Neurobasal A medium (Gibco) supplemented with 2% B27 (Gibco) and 0.5 mM glutamine. Supernatants were collected 48 hrs after transfection and filtered to remove cell debris. Aliquots were flash frozen in liquid nitrogen and stored at −80 °C until use. The viral titer was estimated using the Lenti-X^TM qRT-PCR Titration Kit (Clontech) according to the manufacturer’s instructions. Primary neuronal cultures were transduced with titer-matched lentiviral vectors (1 × 10⁷ copies/dish) that were expected to be sufficient to transduce all of the neurons on the dish.

Effects of the LRAs on HIV transcription were determined by qPCR assay. Jurkat cells were transduced with VSV-G pseudo-typed HIV-1 NL4-3-Luciferase virus for 8 h and treated with compounds (UMB-32, UMB-136, JQ1, or prostratin) for 24 h. Cells were collected and subjected to mRNA extraction (RNeasy® Mini Kit, Qiagen) and reverse transcription (iScript™ cDNA Synthesis Kit, Bio-Rad). The qPCR assays were conducted as previously described with slight modification (Johnston et al., 2009 (link)), using the iTaq™ Universal SYBR® Green Supermix (Bio-Rad) on the CFX Connect™ Real-Time PCR System (Bio-Rad). The following qPCR primers were used: Initiation (10–59 bp) (forward, 5′- GTT AGA CCA GAT CTG AGC CT-3′; reverse, 5′-GTG GGT TCC CTA GTT AGC CA-3′); Elongation (29–180 bp) (forward, 5′-TGG GAG CTC TCT GGC TAACT-3′; reverse, 5′-TGC TAG AGA TTT TCC ACA CTG A-3′).
To determine the effect of UMB-136 on HIV-1 replication, Jurkat cells were infected with full-length HIV-1 NL4-3 (X4) viruses for 8 h before being treated with compounds (UMB-136, JQ1, or prostratin) for 24 h. The medium was collected and subjected to viral RNA extraction, reverse transcription, and qPCR assays by using Lenti-XTM qRT-PCR Titration Kit (Clontech) following the manufacturer's instruction.

Third-generation lentiviral constructs, including pLenti-C-mGFP-P2A-Puro (vector) and human WT TDP-43, were used to generate lentiviruses as previously described [40 (link)]. HEK293T cells were transiently transfected with pLenti-C-mGFP-P2A plasmids, a 3rd generation packaging system, using Lipofectamine 2000 diluted in serum-free CD293 medium (Life Technologies, Carlsbad, CA, USA) to generate lentiviral particles. The cell culture supernatants were collected 48 h later, filtered through a 0.45-mm filter prior to transduction, and particle titers were determined using a Lenti-XTM qRT–PCR titration kit (Clontech, San Francisco, CA, USA). Particles were aliquoted and stored at −80 °C until the experiment. SH-SY5Y cells were infected with GFP-tagged or GFP-tagged human WT TDP-43 (MOI 25) virus particles using 4 μg/mL polybrene (MilliporeSigma). Lentiviral particles were centrifuged at 900× g at 32 °C for 120 min and incubated overnight in a 37 °C CO₂ incubator. The medium was changed to DMEM containing 10% FBS to avoid the toxicity of virus particles. Cells were harvested 2 or 3 days after infection.

The following plasmids were obtained from Addgene including pcDNA3.1-SARS2-Spike mammalian expression plasmid (#145032), NL4-3 mCherry Luciferase dual reporter vector (#44965), and the control env expression vector, VSV-G (pMD2.G) (#12259). The SARS-CoV-2 G614 mutant expression vector was generated as recently reported (Zhang et al., 2020a ). The SARS-CoV-2 pseudotyped virus was produced by transfecting 293T cells with 10 μg of NL4-3 mCherry Luciferase and 10 μg env expression vector: either VSV-G (pMD2.G) or pcDNA3.1-SARS2 Spike using polyethylenimine (PEI) (25 kDa, 1 μg/μL). The virus supernatant was collected at 48 h after transfection and concentrated at 1:100 ratio using Lenti-X^TM Concentrator (TaKaRa/Clontech, 631231). Viral titer was determined using Lenti-X^TM qRT-PCR Titration Kit (TaKaRa/Clontech, 631235).

The lentiviral vectors and ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) were co-transfected into 293 T cells by Trans IT LT-1 (Mirus), and the supernatants were recovered at 48 h post-transfection. The lentivirus titer was determined by using a Lenti-XTM qRT-PCR Titration Kit (Clontech), and the expression levels and AcGFP were determined at 48 h post-inoculation.

Helper plasmids pLP1, pLP2, pLP.VSV.G and backbone plasmid were co-transfected in 293T cells using calcium phosphate (CalPhos Mammalian Transfection Kit, Clontech). After 2 days, lentivirus in supernatants were harvested and concentrated by centrifugation. The viral titer was determined with the Lenti-XTM qRT-PCR Titration Kit (Clontech). Four hours post-plating, HeLa and NIH3T3 cells were infected with serial dilutions of viral particles.

The lentiviral vectors and ViraPower Lentiviral Packaging Mix (Life Technologies) were co-transfected into 293T cells by Trans IT LT-1 (Mirus), and the supernatants were recovered at 48 h post-transfection. The lentivirus titer was determined by using a Lenti XTM qRT-PCR Titration Kit (Clontech), and expression levels and AcGFP were determined at 48 h post-inoculation.