Abstract
Reprogramming of committed cells from one lineage to another is possible in the hematopoietic system using enforced expression of transcription factors. Here we describe methods to convert committed B and T progenitors into macrophages. In order to obtain a labeled population of starting cells, we employ a lineage ancestry system using a cross between lineage-specific Cre recombinase mice and Rosa26 reporter mice. After infection of these well-defined cell populations with a transcription factor containing retroviral vector that also harbors an infection marker, cells are cultured under conditions permissive for both lymphoid and myeloid development. Multicolor flow cytometry is then used to monitor changes in marker expression on the cell surface reflecting changes in cellular identity. These protocols may be modified to trace cellular reprogramming induced by other transcription factors and in other cellular contexts.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Morrison, S.J., N. Uchida, and I.L. Weissman, (1995) The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol, 11: p. 35-71.
Orkin, S.H. and L.I. Zon, (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell, 132(4): p. 631–44.
Orkin, S.H., (2000) Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet, 1(1): p. 57–64.
Sieweke, M.H. and T. Graf, (1998) A transcription factor party during blood cell differentiation. Curr Opin Genet Dev, 8(5): p. 545–51.
Cobaleda, C. and M. Busslinger, (2008) Developmental plasticity of lymphocytes. Curr Opin Immunol, 20(2): p. 139–48.
Graf, T., (2002) Differentiation plasticity of hematopoietic cells. Blood, 99(9): p. 3089–101.
Akashi, K., (2005) Lineage promiscuity and plasticity in hematopoietic development. Ann N Y Acad Sci, 1044: p. 125–31.
Laiosa, C.V., et al., (2006) Reprogramming of committed T cell progenitors to macrophages and dendritic cells by C/EBP alpha and PU.1 transcription factors. Immunity, 25(5): p. 731–44.
Xie, H., et al., (2004) Stepwise reprogramming of B cells into macrophages. Cell, 117(5): p. 663–76.
Ye, M., et al., (2003) Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential. Immunity, 19(5): p. 689–99.
Rickert, R.C., J. Roes, and K. Rajewsky, (1997) B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res, 25(6): p. 1317–8.
Srinivas, S., et al., (2001) Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol, 1: p. 4.
Lee, P.P., et al., (2001) A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity, 15(5): p. 763–74.
Kinsella, T.M. and G.P. Nolan, (1996) Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther, 7(12): p. 1405–13.
Swift, S., et al., (2001) Rapid production of retroviruses for efficient gene delivery to mammalian cells using 293T cell-based systems. Curr Protoc Immunol, Chapter 10 : p. Unit 10 17C.
Collins, L.S. and K. Dorshkind, (1987) A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J Immunol, 138(4): p. 1082–7.
Schmitt, T.M., et al., (2004) Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol, 5(4): p. 410–7.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Xie, H., Laiosa, C.V., Graf, T. (2010). Reprogramming of Committed Lymphoid Cells by Enforced Transcription Factor Expression. In: Ding, S. (eds) Cellular Programming and Reprogramming. Methods in Molecular Biology, vol 636. Humana Press. https://doi.org/10.1007/978-1-60761-691-7_14
Download citation
DOI: https://doi.org/10.1007/978-1-60761-691-7_14
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-690-0
Online ISBN: 978-1-60761-691-7
eBook Packages: Springer Protocols