Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors.
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.
Kutner
Robert H
RH
Gene Therapy Program, Department of Medicine, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA.
Zhang
Xian-Yang
XY
Reiser
Jakob
J
D002460:Cell Line; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011092:Polyethylene Glycols; D016133:Polymerase Chain Reaction; D019076:Transgenes; D014461:Ultracentrifugation; D014776:Virus Cultivation; D014779:Virus Replication
D011092:Polyethylene Glycols