Skip to main content
SpringerLink
Log in

Retroviral Transduction of Quiescent Murine Hematopoietic Stem Cells

  • Protocol
  • First Online:
Book cover Cellular Quiescence

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1686))

Abstract

Hematopoietic stem cells (HSCs) represent an important target cell population in bone marrow transplantation, cell and gene therapy applications, and the development of leukemia models for research. Because the hematopoietic progeny carries the genetic information of HSCs and replenishes the blood and immune system, corrective gene transfer into HSCs provides an ideal therapeutic approach for many monogenic hematological diseases and a useful tool for studies of HSC function and blood formation in normal and malignant hematopoiesis. However, the efficiency of gene transfer into HSCs has been limited by several features of viral vectors, viral titer, methods of viral transduction, and the property of stem cell quiescence. In this chapter, we describe the production of retrovirus using murine stem cell virus (MSCV)-based retroviral vectors and purification and transduction of murine HSCs.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Weissman IL (2000) Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287(5457):1442–1446

    Article  CAS  PubMed  Google Scholar 

  2. Karlsson S (1991) Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 78(10):2481–2492

    CAS  PubMed  Google Scholar 

  3. Chu P, Lutzko C, Stewart AK, Dube ID (1998) Retrovirus-mediated gene transfer into human hematopoietic stem cells. J Mol Med (Berl) 76(3–4):184–192

    Article  CAS  Google Scholar 

  4. Kohn DB, Hershfield MS, Carbonaro D, Shigeoka A, Brooks J, Smogorzewska EM, Barsky LW, Chan R, Burotto F, Annett G, Nolta JA, Crooks G, Kapoor N, Elder M, Wara D, Bowen T, Madsen E, Snyder FF, Bastian J, Muul L, Blaese RM, Weinberg K, Parkman R (1998) T lymphocytes with a normal ADA gene accumulate after transplantation of transduced autologous umbilical cord blood CD34+ cells in ADA-deficient SCID neonates. Nat Med 4(7):775–780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Miller DG, Adam MA, Miller AD (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 10(8):4239–4242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Orlic D, Girard LJ, Jordan CT, Anderson SM, Cline AP, Bodine DM (1996) The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci U S A 93(20):11097–11102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Miller AD, Rosman GJ (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7(9):980–982. 984-986, 989-990

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Grez M, Akgun E, Hilberg F, Ostertag W (1990) Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells. Proc Natl Acad Sci U S A 87(23):9202–9206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Laker C, Meyer J, Schopen A, Friel J, Heberlein C, Ostertag W, Stocking C (1998) Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation. J Virol 72(1):339–348

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Hawley RG, Lieu FH, Fong AZ, Hawley TS (1994) Versatile retroviral vectors for potential use in gene therapy. Gene Ther 1(2):136–138

    CAS  PubMed  Google Scholar 

  11. Hawley RG, Hawley TS, Fong AZ, Quinto C, Collins M, Leonard JP, Goldman SJ (1996) Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11. Proc Natl Acad Sci U S A 93(19):10297–10302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Cherry SR, Biniszkiewicz D, van Parijs L, Baltimore D, Jaenisch R (2000) Retroviral expression in embryonic stem cells and hematopoietic stem cells. Mol Cell Biol 20(20):7419–7426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kume A, Xu R, Ueda Y, Urabe M, Ozawa K (2000) Long-term tracking of murine hematopoietic cells transduced with a bicistronic retrovirus containing CD24 and EGFP genes. Gene Ther 7(14):1193–1199. doi:10.1038/sj.gt.3301225

    Article  CAS  PubMed  Google Scholar 

  14. Sato T, Laver JH, Ogawa M (1999) Reversible expression of CD34 by murine hematopoietic stem cells. Blood 94(8):2548–2554

    CAS  PubMed  Google Scholar 

  15. Randall TD, Weissman IL (1997) Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 89(10):3596–3606

    CAS  PubMed  Google Scholar 

  16. Busch K, Klapproth K, Barile M, Flossdorf M, Holland-Letz T, Schlenner SM, Reth M, Hofer T, Rodewald HR (2015) Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518(7540):542–546. doi:10.1038/nature14242

    Article  CAS  PubMed  Google Scholar 

  17. Hanenberg H, Xiao XL, Dilloo D, Hashino K, Kato I, Williams DA (1996) Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 2(8):876–882

    Article  CAS  PubMed  Google Scholar 

  18. Chono H, Yoshioka H, Ueno M, Kato I (2001) Removal of inhibitory substances with recombinant fibronectin-CH-296 plates enhances the retroviral transduction efficiency of CD34(+)CD38(−) bone marrow cells. J Biochem 130(3):331–334

    Article  CAS  PubMed  Google Scholar 

  19. Hanenberg H, Hashino K, Konishi H, Hock RA, Kato I, Williams DA (1997) Optimization of fibronectin-assisted retroviral gene transfer into human CD34+ hematopoietic cells. Hum Gene Ther 8(18):2193–2206. doi:10.1089/hum.1997.8.18-2193

    Article  CAS  PubMed  Google Scholar 

  20. Tonks A, Tonks AJ, Pearn L, Mohamad Z, Burnett AK, Darley RL (2005) Optimized retroviral transduction protocol which preserves the primitive subpopulation of human hematopoietic cells. Biotechnol Prog 21(3):953–958. doi:10.1021/bp0500314

    Article  CAS  PubMed  Google Scholar 

  21. Zhou P, Lee J, Moore P, Brasky KM (2001) High-efficiency gene transfer into rhesus macaque primary T lymphocytes by combining 32 degrees C centrifugation and CH-296-coated plates: effect of gene transfer protocol on T cell homing receptor expression. Hum Gene Ther 12(15):1843–1855. doi:10.1089/104303401753153901

    Article  CAS  PubMed  Google Scholar 

  22. Quintas-Cardama A, Yeh RK, Hollyman D, Stefanski J, Taylor C, Nikhamin Y, Imperato G, Sadelain M, Riviere I, Brentjens RJ (2007) Multifactorial optimization of gammaretroviral gene transfer into human T lymphocytes for clinical application. Hum Gene Ther 18(12):1253–1260. doi:10.1089/hum.2007.088

    Article  CAS  PubMed  Google Scholar 

  23. Jensen TW, Chen Y, Miller WM (2003) Small increases in pH enhance retroviral vector transduction efficiency of NIH-3T3 cells. Biotechnol Prog 19(1):216–223. doi:10.1021/bp025604g

    Article  CAS  PubMed  Google Scholar 

  24. Swaney WP, Sorgi FL, Bahnson AB, Barranger JA (1997) The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer. Gene Ther 4(12):1379–1386. doi:10.1038/sj.gt.3300529

    Article  CAS  PubMed  Google Scholar 

  25. Davis HE, Morgan JR, Yarmush ML (2002) Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97(2–3):159–172

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank members of the laboratory for helpful discussions. Support for this work was provided in part by the Cancer Prevention Research Institute of Texas to H.D.L. (RP140179) and the National Cancer Institute to H.D.L. (RO1 CA207086-01A1).

Author information

Authors and Affiliations

  1. Department of Pathology and Immunology, Baylor College of Medicine, Texas Children’s Hospital, 1102 Bates Street, FC830.20, Houston, TX, 77030, USA

    Chun Shik Park & H. Daniel Lacorazza

Authors
  1. Chun Shik Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Daniel Lacorazza
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H. Daniel Lacorazza .

Editor information

Editors and Affiliations

  1. Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA

    H. Daniel Lacorazza

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Park, C.S., Lacorazza, H.D. (2018). Retroviral Transduction of Quiescent Murine Hematopoietic Stem Cells. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7371-2_13

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7370-5

  • Online ISBN: 978-1-4939-7371-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation