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      Characterisation of a stably integrated expression system for exogenous protein expression in DT40.

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          Abstract

          The use of constitutive promoters to drive exogenous protein expression is an important tool for the study of diverse biological processes. To create and characterise a stably integrated expression system for DT40 cells, we constructed integration cassettes for three commonly used promoter elements; CMV, CBA or CAG, and used these to stably integrate a TOPBP1 transgene at the OVA locus, a transcriptionally silent locus commonly used in DT40. We next performed a comparative analysis of protein expression levels and identified CAG as the most efficient of the promoter elements we have tested in DT40 cells. To assess whether the site of integration affected the levels of transgene expression, a second chromosomal locus, immediately adjacent to the endogenous TOPBP1 gene, was tested for CAG. No major differences in TopBP1 overexpression were observed. This confirms that use of the OVA locus for integrating transgenes is a rational choice for DT40. Finally, we demonstrate that our stably integrated overexpression system (SIOS) constructs can be efficiently excised by the induction of tamoxifen-regulated Cre expression. Taken together, SIOS is an easy-to-use and versatile system for constitutive, reversible exogenous protein production that provides a range of potential expression levels. This will be a useful experimental tool for future DT40 experiments.

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          Author and article information

          Journal
          Wellcome Open Res
          Wellcome open research
          F1000 Research, Ltd.
          2398-502X
          2398-502X
          2017
          : 2
          Affiliations
          [1 ] Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, BN1 9RQ, UK.
          Article
          10.12688/wellcomeopenres.11816.2
          5482329
          28695215
          c3f18312-46a5-463e-8567-5cfede7e9e6d
          History

          DT40,promoter,protein expression,transcription
          DT40, promoter, protein expression, transcription

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