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Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis

Abstract

Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >106 per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

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Acknowledgements

We are grateful to Sabine Helbing for excellent technical assistance, Claudia Lange for providing MSC and Carol Stocking for critical reading of the paper. We thank many colleagues for their kind support with various cells and constructs: Luk van Parijs (pLentiLox3.7), Roger Y Tsien (tdTomato and mCherry cDNA), Atsushi Miyawaki/ Timm Schroeder (Venus cDNA), David W Piston (Cerulean cDNA), and David H Rowitch (murine Olig2 cDNA). This work was supported by the Deutsche Forschungsgemeinschaft (FE568/5-2; FE568/11–1 to BF), INTAS (06–1000013–9117 to BF), the Bundesministerium für Bildung und Forschung (BMBF 01GN0501 to UB), and the Frankfurter Stiftung für krebskranke Kinder. This report is part of the doctoral thesis of Kristoffer Weber.

Author contributions: KW conception and design, collection and/or assembly of data, data analysis and interpretation, and final approval of paper; UM collection and/or assembly of data and final approval of paper; BP collection and/or assembly of data and final approval of paper; UB collection and/or assembly of data and final approval of paper; and BF conception and design, data analysis and interpretation and paper writing.

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Correspondence to B Fehse.

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Weber, K., Mock, U., Petrowitz, B. et al. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis. Gene Ther 17, 511–520 (2010). https://doi.org/10.1038/gt.2009.149

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  • DOI: https://doi.org/10.1038/gt.2009.149

Keywords

  • lentiviral vectors
  • gene ontology
  • fluorescent protein
  • antibiotic resistance
  • selection markers
  • functional gene analysis

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