The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.
Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.
Coroadinha
A S
AS
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica (IBET/ITQB), Oeiras, Portugal.
Schucht
R
R
Gama-Norton
L
L
Wirth
D
D
Hauser
H
H
Carrondo
M J T
MJ
D002460:Cell Line / Q000821:virology*; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase