Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene.
Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.
Loew
Rainer
R
EUFETS AG, Vollmersbachstrasse 66, D-55743 Idar-Oberstein, Germany. rainer.loew@fresenius-biotech.com
Selevsek
Nathalie
N
Fehse
Boris
B
von Laer
Dorothee
D
Baum
Christopher
C
Fauser
Axel
A
Kuehlcke
Klaus
K
D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D018952:Antigens, CD34 / Q000235:genetics; D018929:Cell Culture Techniques; D015870:Gene Expression; D015321:Gene Rearrangement / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics* / Q000627:therapeutic use; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D011533:Proviruses / Q000235:genetics / Q000254:growth & development; D011995:Recombination, Genetic / Q000235:genetics; D012190:Retroviridae / Q000235:genetics* / Q000254:growth & development; D020079:Terminal Repeat Sequences / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D018952:Antigens, CD34; C497857:CD46 protein, human; D005819:Genetic Markers; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases