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

Blood Cells Mol Dis. 1998 Jun;24(2):167-82. doi: 10.1006/bcmd.1998.0184.


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 high multiplicity of infection (MOI). Each cell in the resulting polyclonal population of producer cells contained multiple copies of the unrearranged vector genome. Greater than 95% of the clones derived from this population produced vector particles as judged by slot blot analysis of viral RNA from conditioned media. Numerous clones with estimated titers of 10(5)-10(6) were identified. These titers were confirmed using a standard vector genome transmission assay. This approach significantly enhances the ability, without large scale screening, to easily identify high-titer clones lacking a selectable marker and should facilitate the routine use of simplified gene marking and therapeutic vectors.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells / virology
  • Animals
  • COS Cells / virology
  • Cell Line / virology
  • Cell Separation
  • Cloning, Molecular / methods
  • Flow Cytometry
  • Genetic Markers*
  • Genetic Vectors / genetics*
  • Genetic Vectors / physiology
  • Kidney
  • Mice
  • Retroviridae / genetics*
  • Retroviridae / physiology
  • Retroviridae Proteins, Oncogenic / analysis
  • Selection, Genetic
  • Transfection
  • Viral Envelope Proteins / analysis
  • Virus Cultivation / methods*
  • Virus Replication


  • Genetic Markers
  • Retroviridae Proteins, Oncogenic
  • Viral Envelope Proteins