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Session IV – Chromosome Engineering

Visible transient expression of EGFP requires intranuclear injection of large copy numbers

Gene Therapy volume 9, pages 727–730 (2002)Cite this article

Abstract

For the development of human artificial chromosomes (HACs) as a gene transfer vehicle we need to assess the efficiency of de novo chromosome formation depending on the type and the copy number of transferred DNA constructs. In order to check transient EGFP expression as a reporter to immediately detect presence of transfected DNA, we microinjected approximately 1 to 105 copies of pEGFP-N1 plasmid into the nucleus of various cell types. Whether using primary, immortalized, or tumor cells, at least 103–104 copies were required to generate a visible green signal in the majority of the 50–90% of cells surviving injection. Generally, the cells showed relatively constant, copy number-dependent signals. 103 copies resulted in faint and 105 in bright fluorescence under the microscope. In addition, the different copy number groups contained a small fraction of cells showing much stronger fluorescence, indicating activation or lack of suppression which facilitates detection of as few as 102 transferred copies in rare instances. Thus, transient expression from single copies is not sufficient to reliably detect presence of DNA in the nucleus. The result is relevant for the development of low copy HAC transfer protocols.

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Acknowledgements

We thank Jim Lausier and Josef Rosenecker for help with immunostaining and access to imaging equippment, Dieter Gruenert and the University of Califonia for providing a batch of 16HBE14o−, Brenda Grimes for HT1080 cells, Konstanze Hörtnagel for sparing batches of human fibroblasts, and Thomas Meitinger for his support. The work was supported by grants to DS from the Deutsche Forschungsgemeinschaft (DFG), the Friedrich Baur Stiftung, and the Forschungsgemeinschaft Mukoviszidose.

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Authors and Affiliations

  1. Institute of Human Genetics, Technical University, Munich, Germany

    D Schindelhauer

  2. Department of Medical Genetics, Kinderpoliklinik, Ludwig-Maximilians-University, Munich, Germany

    D Schindelhauer & A Laner

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  1. D Schindelhauer
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  2. A Laner
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Schindelhauer, D., Laner, A. Visible transient expression of EGFP requires intranuclear injection of large copy numbers. Gene Ther 9, 727–730 (2002). https://doi.org/10.1038/sj.gt.3301755

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