Abstract
The ability to efficiently transduce hematopoietic stem cells (HSC) represents a powerful methodology by which to study the role of specific genes on HSC function, as well as to broaden the potential of gene therapy for hematopoietic related diseases. While retroviruses have been used extensively to transduce a variety of cell types, HIV-derived lentiviruses prove superior for transduction of quiescent HSC due to their ability to infect non-dividing cells. Quality of lentiviral supernatants and starting cells are vital to obtain reproducible consistent results, and therefore, here we describe the production of concentrated lentiviral preparations, the purification of HSC from total mouse bone marrow, and their transduction to obtain long-term HSC engraftment with persistent gene transfer and expression of the desired transgene.
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References
Kohn DB, Sadelain M, Dunbar C et al (2003) American Society of Gene Therapy (ASGT) ad hoc subcommittee on retroviral-mediated gene transfer to hematopoietic stem cells. Mol Ther 8(2):180–187
Maetzig T, Galla M, Baum C et al (2011) Gammaretroviral vectors: biology, technology and application. Viruses 3(6):677–713
Somia N, Verma IM (2000) Gene therapy: trials and tribulations. Nat Rev Genet 1(2):91–99
Peters SO, Kittler EL, Ramshaw HS et al (1996) Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 87(1):30–37
Yonemura Y, Ku H, Hirayama F et al (1996) Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc Natl Acad Sci U S A 93(9):4040–4044
Blomer U, Naldini L, Kafri T et al (1997) Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J Virol 71(9):6641–6649
Kafri T, Blomer U, Peterson DA et al (1997) Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 17(3):314–317
Naldini L, Blomer U, Gallay P et al (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267
Uchida N, Sutton RE, Friera AM et al (1998) HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc Natl Acad Sci U S A 95(20):11939–11944
Heckl D, Wicke DC, Brugman MH et al (2011) Lentiviral gene transfer regenerates hematopoietic stem cells in a mouse model for Mpl-deficient aplastic anemia. Blood 117(14):3737–3747
Cavazzana-Calvo M, Payen E, Negre O et al (2010) Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 467(7313):318–322
Sakuma T, Barry MA, Ikeda Y (2012) Lentiviral vectors: basic to translational. Biochem J 443(3):603–618
Mostoslavsky G, Kotton DN, Fabian AJ et al (2005) Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation. Mol Ther 11(6):932–940
Mostoslavsky G, Fabian AJ, Rooney S et al (2006) Complete correction of murine Artemis immunodeficiency by lentiviral vector-mediated gene transfer. Proc Natl Acad Sci U S A 103(44):16406–16411
Baum C, Dullmann J, Li Z et al (2003) Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 101(6):2099–2114
Hacein-Bey-Abina S, von Kalle C, Schmidt M et al (2003) A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 348(3):255–256
Goodell MA, Brose K, Paradis G et al (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183(4):1797–1806
Zhou S, Schuetz JD, Bunting KD et al (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7(9):1028–1034
Ema H, Takano H, Sudo K et al (2000) In vitro self-renewal division of hematopoietic stem cells. J Exp Med 192(9):1281–1288
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Thomas, D., Mostoslavsky, G. (2014). Efficient Transduction of Hematopoietic Stem Cells and Its Potential for Gene Correction of Hematopoietic Diseases. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_29
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DOI: https://doi.org/10.1007/978-1-62703-761-7_29
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Publisher Name: Humana Press, Totowa, NJ
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