Browsing Publications of Dept. Gene Regulation and Differentiation (RDIF) by Subjects
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Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
Retroviral vector performance in defined chromosomal Loci of modular packaging cell lines.The improvement of safety and titer of retroviral vectors produced in standard retroviral packaging cell lines is hampered because production relies on uncontrollable vector integration events. The influences of chromosomal surroundings make it difficult to dissect the performance of a specific vector from the chromosomal surroundings of the respective integration site. Taking advantage of a technology that relies on the use of packaging cell lines with predefined integration sites, we have systematically evaluated the performance of several retroviral vectors. In two previously established modular packaging cell lines (Flp293A and 293 FLEX) with single, defined chromosomal integration sites, retroviral vectors were integrated by means of Flp-mediated site-specific recombination. Vectors that are distinguished by different long terminal repeat promoters were introduced in either the sense or reverse orientation. The results show that the promoter, viral vector orientation, and integration site are the main determinants of the titer. Furthermore, we exploited the viral production systems to evaluate read-through activity. Read-through is thought to be caused by inefficient termination of vector transcription and is inherent to the nature of retroviral vectors. We assessed the frequency of transduction of sequences flanking the retroviral vectors from both integration sites. The approach presented here provides a platform for systematic design and evaluation of the efficiency and safety of retroviral vectors optimized for a given producer cell line.