Protocol

Generation of Stable Vector-Producing Cells for Retroviral Vectors

This protocol was adapted from "Retroviral Vectors," Chapter 2, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

This procedure describes the generation of clonal vector-producing cells that will provide an unlimited amount of unrearranged retroviral vector. The procedure involves transfection of one packaging cell line to generate a vector that is used to transduce a second packaging cell line. The resultant vector-producing clones generally contain a single integrated copy of the retroviral vector, and virus produced from this integrated vector is as genetically homogeneous as possible. Although the vector produced by a given packaging cell line can sometimes be used to transduce the same cell line, the transduction rate is typically low because of receptor blockage by the Env protein made by the target packaging cells. Indeed, this procedure will select for target cells that express low Env protein levels and thus are less resistant to transduction, but at the same time will ultimately produce less vector because of low Env production. Therefore, to obtain the highest vector titers, it is important to use pairs of packaging cells such that receptor blockage is not an issue. In this example, we use PE501 ecotropic packaging cells for transfection and broad-host-range PT67 packaging cells to make stable vector-producing cells.