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- W2080247247 abstract "▼The isolation of recombinant mammalian cells with defined expression levels requires extensive screening. Due to random integration, transfection or injection of DNA leads to cell clones in which the integration site and multicopy number affects foreign gene expression. To overcome these problems efforts have been made to create cell lines with predictable gene expression. One approach is to target the expression cassette to predefined chromosomal loci in the cell line of interest. FRT or loxP-tagged reporter genes, introduced into favorable chromosomal loci, permit analysis of cells with relevant expression. Subsequently, FLPor Crerecombinase enable sequence-specific targeting of these loci. However, the FRT/Flpand loxP/Crerecombinase systems still demand screening for specific integration events (Ref. 1, 2, 3). By contrast with embryonic stem cells, homologous and sequence specific recombination in fibroblastoid cells is considered to be very inefficient. While for embryonic stem cells a protocol has recently been published that allows efficient targeting using the FRT/Flp system (Ref. 4), in fibroblastoid cells the FRT/Flp system was not considered favorable (Ref. 5). Here, we present an improved targeting method that allows the isolation of targeted recombinants in one step in fibroblastoid cells. Cell lines containing a FRTtagged retroviral reporter construct can be targeted with the gene of interest with 100% efficiency. The strength of this system lies in the unique combination of the characteristics of retroviruses, the improved double FRT/Flp system and a stringent selection for the targeting event. In our system a retroviral vector pCMVGALEO is used to screen for appropriate integration sites. The retroviral vector pCMVGALEO contains β-galactosidase (LacZ), followed by the fusion gene hygromycin B phosphotransferase/thymidine kinase as a positive/negative selection marker. The 3′ LTR of pCMVGALEO contains two FRT-sites in tandem, a wild type (F1) and a mutant FRT form (F2)," @default.
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- W2080247247 date "1998-01-01" @default.
- W2080247247 modified "2023-10-18" @default.
- W2080247247 title "Efficient targeting of retrovirally FRT-tagged chromosomal loci" @default.
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- W2080247247 doi "https://doi.org/10.1016/s1366-2120(08)70118-1" @default.
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