An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene

Blood Cells, Molecules & Diseases
D A PersonsE F Vanin

Abstract

Most retroviral vectors used in preclinical and clinical studies contain a selectable marker gene to facilitate the generation of producer clones. However, the expression of such genes in target cells is often undesirable since this may modify cellular phenotype and invoke a host immune response. Unfortunately, the efficient identification of high-titer producer clones for vectors lacking a selectable marker gene continues to be problematic and lacking for a standard methodology. Despite recent improvements in the screening techniques for identifying high-titer producer clones without the aid of a selectable marker, a solution to the fundamental problem of the very low frequency occurrence of high-titer clones within the starting cell population has not emerged. We have developed a strategy which greatly increases the frequency of virus-producing clones, including those with high-titer, within the population of transduced cells to be screened. This approach relies on the use of high-titer vector preparations generated in 293T cells by co-transfection of retroviral packaging and vector plasmids. Viral preparations of a vector lacking a selectable marker were used to repeatedly transduce exponentially growing packaging cells at a...Continue Reading

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