title: Stability and homogeneity of transgene expression in isogenic cells.
abstract: Genetically engineered cells are an important tool not only for basic research applications, but also for biotechnology and molecular medicine. Among the issues yet to be solved for this technology are the consequences of randomly integrating DNA into a cell's genome. The problems encountered range from unpredictable expression levels to safety concerns. Recombinase-mediated chromosome engineering is a popular tool for generating stably transfected isogenic cell lines. Using this approach, single-copy integration of foreign DNA fragments can be achieved at predetermined chromosomal loci in the genome. We used such a technology based on the Flp/Flp recombinase target (FRT) recombination system in human 293 cells for comparative promoter studies. The expected integration patterns were obtained with high frequency. In contrast, the phenotypic characterization of expression of the integrated reporter transgene showed remarkable differences between isogenic cell lines, ranging from homogenous expression to mosaic to even complete expression silencing. As long as cell clones with homogenous expression were kept under selective conditions, their expression characteristics could be maintained over a long period. However, this desirable phenotype was progressively lost upon withdrawal of selective pressure. These results were also reflected by the expression instability of the reporter cassette originally inserted in the recipient cell line. Thus, selection for appropriate integration events that ensure reproducible and long-term gene expression has to go beyond the verification of isogenicity.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany.
authors.forename: Weimin
authors.initials: W
authors.lastname: Liu
authors.forename: Yuanzhu
authors.initials: Y
authors.lastname: Xiong
authors.forename: Manfred
authors.initials: M
authors.lastname: Gossen
mesh_terms: D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D019076:Transgenes
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase