Lentiviral Transduction

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Enhancing Lentiviral Transduction Efficiency

Methods for Enhancing Lentiviral Transduction Efficiency

Abstract

An experiment to directly compare three methods of lentiviral transduction of Jurkat cells was conducted in order to determine the method that yields the greatest transduction efficiency. Spinoculation was carried out in parallel with an overnight incubation of virus with cells in the presence of polybrene (hexadimethrine bromide), and with transductions conducted on fibronection-coated plates. Spinoculation was shown to be the most successful method of transduction for Jurkat cells.

Introduction

RNAi is a useful tool for functional analysis of genes and developing a potential therapeutic strategy for various diseases. Unlike murinebased MMLV or MSCV retroviral systems, lentiviral-based particles permit efficient transduction and integration of a specific shRNA construct into differentiated and non-dividing cells.1 While incubation of lentivirus pseudotyped with G glycoprotein from vesicular stomatitis virus (VSV-G) and cells in the presence of polybrene can efficiently serve as the method of transduction for many cell types, some cells are more difficult to transduce. Therefore, modifications have been made to transduction protocols for hardto- transduce lines, such as T-cells, to facilitate binding of viral envelope protein to cells. Some of these modifications include spinoculation, magnetic transduction (see MISSION^® ExpressMag^® product line), transduction of cells on fibronectin-coated plates, and incubation of cells with concentrated viral particles4 (greater than or equal to 108 TU/ mL). The following is a spinoculation protocol that was successful in the transduction of VSV-G pseudotyped lentivirus in Jurkat cells. The protocol was carried out in parallel with an overnight incubation of virus with cells in the presence of polybrene and with transductions conducted on fibronection-coated plates.