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Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

Journal of Biotechnology

Animals, Cell Differentiation, genetics, Cell Line, Chromosome Mapping, methods, Epithelial Cells, physiology, Gene Expression Profiling, Gene Targeting, Gene Transfer Techniques, Rats, Recombinant Proteins, biosynthesis

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      Abstract

      Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

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      15664078
      10.1016/j.jbiotec.2004.10.014

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