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Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

Gene Therapy volume 17, pages 14–25 (2010)Cite this article

Abstract

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated ‘human immune system’ (HIS) mice by transplanting newborn BALB/c Rag2−/−IL-2Rγc−/− immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.

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Acknowledgements

This research was sponsored by the Dutch AIDS Foundation (AIDS Fonds, Grant 2005022), the International AIDS Vaccine Initiative (IAVI), the Technology Foundation STW (the applied science division of NWO and the technology program of the Ministry of Economic Affairs, Utrecht, The Netherlands) and the NIH/NIAD (No. R21-AI073136). M Centlivre is supported by Marie Curie Intra-European fellowship (MEIF-CT-2007-039689). N Legrand is supported by the Bill and Melinda Gates Foundation (Grand Challenges in Global Health program—GC4 ‘Human Vaccine Consortium’). We thank the staff of the ABSL-3 unit of the Animal Research Institute Amsterdam (ARIA) of the AMC-UvA for excellent care of the animals. We also thank Joost Dalhuizen for technical support, Berend Hooibrink for expertise in cell sorting and maintenance of the flow cytometry facility and Marc Douaisi and Christel Uittenbogaart for discussions. Finally, we are grateful to the Bloemenhove Clinic (Heemstede, The Netherlands) for providing fetal tissues. MC designed and performed the research, collected, analyzed and interpreted the data, and wrote the paper. XZ contributed new reagents, and analyzed and interpreted the data. SP and KW performed the research. WK and ATD contributed new reagents. BBl, JS and BBe analyzed and interpreted the data. NL designed and performed the research; collected, analyzed and interpreted the data, and wrote the paper. All authors read and edited the paper.

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Author notes

  1. S M Pouw, K Weijer and N Legrand: Part of the Human Vaccine Consortium, ‘Grand Challenge in Global Health #4: devise reliable testing systems for new vaccines’, http://www.hv-consortium.org

Authors and Affiliations

  1. Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam (AMC-UvA), Amsterdam, The Netherlands

    M Centlivre, X Zhou, W Kleibeuker, A T Das & B Berkhout

  2. Department of Cell Biology and Histology, Center for Immunology of Amsterdam (CIA), Academic Medical Center of the University of Amsterdam (AMC-UvA), Amsterdam, The Netherlands

    S M Pouw, K Weijer, B Blom & N Legrand

  3. Department of Experimental Hepatology, Liver Center, Academic Medical Center of the University of Amsterdam (AMC-UvA), Amsterdam, The Netherlands

    J Seppen

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Correspondence to N Legrand.

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Centlivre, M., Zhou, X., Pouw, S. et al. Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo. Gene Ther 17, 14–25 (2010). https://doi.org/10.1038/gt.2009.109

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