Get Pure High-Titer Virus with Every Packaging Prep with LentiFuge™

Most experiments that rely on lentiviral technology to deliver genetic elements benefit greatly by starting with pure high-titer virus. With some easy-to-transduce cell systems grown in routine medium, such as DMEM, it may be possible to squeak by with a dilute viral supernatant containing proteins and factors from the packaging cell culture. However, protocols involving cells that require different media (esp. cells that grow in suspension) or cells that are difficult-to-transduce (stem cells, blood cells, etc.) will likely require viral concentrations 100-fold more dense than just the supernatant. Freezing small, highly-concentrated aliquots is much more efficient also.

The problem, of course, has always been how to concentrate the virus without concentrating the exogeneous proteins and other factors present in the supernatant. The traditional approach usually involves either an inconvenient and time-consuming sucrose-gradient ultracentrifugation or a messy PEG precipitation followed by centrifugation which co-precipitates most of the proteins and other impurities in the cells and media.

Fortunately, our experience over the years manipulating lentiviruses has lead to a solution — LentiFuge™ Viral Concentration Reagent. It aggregates viral particles together so they can be isolated and separated from other impurities in the medium by standard high-speed centrifugation. The whole LentiFuge isolation protocol takes just 2 hours. One hour incubation with the Reagent, and a second hour for centrifugation in a Beckman JA-14, JA-10, or similar rotor. After these two short steps, the virus is simply resuspended and either used immediately or aliquoted and frozen.

Data from a recent experiment (below) shows that LentiFuge not only concentrates the virus by 2-fold, but it also removes impurities from the viral preparation. The figure shows the transduction efficiency ratio of concentrated virus versus pre-concentrated virus. With LentiFuge, essentially all the virus was recovered and the LentiFuge Reagent gave much better yields than the more traditional PEG protocol.

Viral supernatant was collected at 48-hrs post-transfection concentrated using either 1) high-speed centrifugation, 2) LentiFuge treatment with centrifugation, 3) PEG (polyethylene glycol) treatment with centrifugation, or 4) not concentrated. The resulting concentrated virus was used to transduce cells in culture.

LentiFuge concentration results in a higher yield and virus purity compared with conventional methods

For more information or to purchase LentiFuge, click here.

Leave a comment

Comments will be approved before showing up.

Also in Cellecta Blog & News

Inducible Cas9 Expression in a Single Lentiviral Vector

Introducing Inducible Cas9 Expression in a Single Lentiviral Vector to make cells capable of high Cas9 expression for a limited time during which CRISPR-mediated targeted rearrangements can occur, and then shut off Cas9 expression for downstream assays with the modified cells.
Read More
Insertion of 10X Genomics' Capture Sequences Does Not Affect HEAT-Tracr sgRNA Efficacy

Perturb-Seq or CROP-Seq screens make use of single-cell RNA-Sequencing in conjunction with a pooled CRISPR library to identify transcriptional changes and, by implication, activation or deactivation of cellular pathways related to phenotypic changes produced by specific sgRNA-mediated gene knockouts.
Read More
Core Population of Cancer Stem Cells Mediates Therapeutic Resistance in Tumors

Researchers at MD Anderson Cancer Center recently used a Cellecta CloneTracker Barcode Library to label patient-derived xenograft (PDX) cells and establish a stable population of aggressive tumorigenic cells with a specific set of barcodes. With this population of barcoded tumorigenic clones, the investigators...
Read More