Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors

Abstract

Over the past decade, lentiviral vectors have emerged as powerful tools for transgene delivery. The use of lentiviral vectors has become commonplace and applications in the fields of neuroscience, hematology, developmental biology, stem cell biology and transgenesis are rapidly emerging. Also, lentiviral vectors are at present being explored in the context of human clinical trials. Here we describe improved protocols to generate highly concentrated lentiviral vector pseudotypes involving different envelope glycoproteins. In this protocol, vector stocks are prepared by transient transfection using standard cell culture media or serum-free media. Such stocks are then concentrated by ultracentrifugation and/or ion exchange chromatography, or by precipitation using polyethylene glycol 6000, resulting in vector titers of up to 10(10) transducing units per milliliter and above. We also provide reliable real-time PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells or on vector RNA. These production/concentration methods result in high-titer vector preparations that show reduced toxicity compared with lentiviral vectors produced using standard protocols involving ultracentrifugation-based methods. The vector production and titration protocol described here can be completed within 8 d.

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…For lentiviral vector production, EGFP expression plasmid (pEGIP) was purchased from Addgene (29777) 20 . pEGIP plasmid was packaged and purified as a lentiviral vector according to published papers and the vesicular stomatitis virus Env glycoprotein (VSV-G) is typically used 41 . Non-integrated lentivirus was packaged by pLV-HELP-NIL and pLViVSV-G (Invivogen, San Diego, CA), according to the manufacturer's instructions.…”

Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells

“…Physical particle titers were determined by measuring HIV-1 p24 capsid contents using a commercial ELISA kit (PerkinElmer Life Sciences). Infectious titers were determined on HCT116 cells using either the detection of eGFP by flow cytometry (FACSCalibur, BD Biosciences) with titers expressed as transducing units per milliliter (35) or using quantitative PCR with titers expressed as infectious genome per milliliter (34).…”

SOCS3 Modulates the Response to Enzalutamide and Is Regulated by Androgen Receptor Signaling and CpG Methylation in Prostate Cancer Cells

“…For production of lentiviral particles, HEK293FT cells were transfected with psPAX2, pVSV-G, and the corresponding pLenti6.4 expression vector using the calcium phosphate transfection method (35). Viral supernatants were harvested 48 hours after transfection and passed through a 0.45-mm filter.…”