Transient Transfection Methods for Preparation of High‐Titer Retroviral Supernatants

Abstract

Generation of high-titer retrovirus by transient production not only is less laborious than production of stable retroviral producer cell lines, but also has allowed the production of high-titer retroviral supernatants from cDNAs that cannot be achieved by stable producer cell lines. Transient transfection has also increased the versatility of retrovirus-mediated gene transfer to include the rapid testing of different constructs, viral pseudotyping, and construction of retroviral cDNA libraries. Systems based on human 293 cells, an adenovirus-transformed human embryonic kidney cell line have produced the highest retroviral titers and are the most widely used. This unit describes methods for optimizing retroviral production from the 293-based systems and for growing and freezing 293 cells. Methods are included for pseudotyping the virus with VSV G protein by sequential transfection or cotransfection. Virus produced by transiently transfected cells can be used to infect cells. Protocols are provided for infection of adherent cells either directly with retroviral supernatant or by spin infection. In addition, procedures are included for infection of nonadherent cells by addition of retrovirus supernatant, cocultivation with producer cells, or spin infection. These infection methods are also applicable to retrovirus produced by any of the stable producer cell lines.