A convenient method for positive selection of retroviral producing cells generating vectors devoid of selectable markers.
Early retroviral vectors containing both a therapeutic gene and a dominant selectable marker gene, offered some distinct advantages with respect to gene therapy, in that they simplified the generation, isolation, and titration of retroviral producer cell clones, as well as the evaluation and selection of successfully targeted cells. However, a number of problems were engendered by this strategy: the promoter driving the selectable marker gene could interfere with transcription of the therapeutic gene, and immune responses could be induced to cells expressing foreign proteins of selection marker origin. Simplified retroviral vectors, which lack a selection marker gene, were constructed to address these problems, but the inability to use a selection marker has made identification and cloning of virus producing transfected cells a heavy burden. To maintain the benefits of simplified retroviral vectors, while providing a facile means to select packaging cells transfected with retroviral DNA, we cloned the bacterial selection marker gene encoding neomycin phosphotransferase (neo) into the plasmid backbone of the vector, but outside of the provirus, resulting in efficient selection of transfected packaging cells and generation of packaged virus which lacks the neo gene. This novel approach generates greater numbers of high infectious titer producing clones, after selection in G418 media, than does a co-transfection approach, due to integration of higher construct copy numbers per cell. No transmission of the selection marker gene to target cells was observed following retroviral transduction. Thus, our strategy eliminates the adverse consequences of a selection-based method, while diminishing the burden of identification of packaging cells transfected with vectors devoid of selectable markers.
Xu
Lai
L
Division of Therapeutic Proteins, CDER, FDA, Building 29A, Room 2B-10, 29 Lincoln Dr., Bethesda, MD 20892, USA.
Tsuji
Kazuhide
K
Mostowski
Howard
H
Otsu
Makoto
M
Candotti
Fabio
F
Rosenberg
Amy S
AS
D004279:DNA, Viral / Q000235:genetics; D005819:Genetic Markers; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D019868:Kanamycin Kinase / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D014773:Virology / Q000379:methods; D014779:Virus Replication
D004279:DNA, Viral; D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase