An improved method for generating retroviral producer clones for vectors lacking a selectable marker gene.
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.
Persons
D A
DA
Department of Hematology/Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Mehaffey
M G
MG
Kaleko
M
M
Nienhuis
A W
AW
Vanin
E F
EF
D016475:3T3 Cells / Q000821:virology; D000818:Animals; D019556:COS Cells / Q000821:virology; D002460:Cell Line / Q000821:virology; D002469:Cell Separation; D003001:Cloning, Molecular / Q000379:methods; D005434:Flow Cytometry; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics* / Q000502:physiology; D007668:Kidney; D051379:Mice; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D015682:Retroviridae Proteins, Oncogenic / Q000032:analysis; D012641:Selection, Genetic; D014162:Transfection; D014759:Viral Envelope Proteins / Q000032:analysis; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication
D005819:Genetic Markers; D015682:Retroviridae Proteins, Oncogenic; D014759:Viral Envelope Proteins