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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2637-2646
Genetic Modification of Human B-Cell Development: B-Cell
Development Is Inhibited by the Dominant Negative Helix Loop Helix
Factor Id3
Ana C. Jaleco,
Alexander P.A. Stegmann,
Mirjam H.M. Heemskerk,
Franka Couwenberg,
Arjen Q. Bakker,
Kees Weijer, and
Hergen Spits
From the Division of Immunology, The Netherlands Cancer Institute,
Antoni van Leeuwenhoek Huis, Amsterdam, The Netherlands.
Transgenic and gene targeted mice have contributed greatly to our
understanding of the mechanisms underlying B-cell development. We
describe here a model system that allows us to apply molecular genetic
techniques to the analysis of human B-cell development. We constructed
a retroviral vector with a multiple cloning site connected to a gene
encoding green fluorescent protein by an internal ribosomal entry site.
Human CD34+CD38 fetal liver cells,
cultured overnight in a combination of stem cell factor and
interleukin-7 (IL-7), could be transduced with 30% efficiency. We
ligated the gene encoding the dominant negative helix loop helix (HLH)
factor Id3 that inhibits many enhancing basic HLH transcription factors
into this vector. CD34+CD38 FL cells were
transduced with Id3-IRES-GFP and cultured with the murine stromal cell
line S17. In addition, we cultured the transduced cells in a
reaggregate culture system with an SV-transformed human fibroblast cell
line (SV19). It was observed that overexpression of Id3 inhibited
development of B cells in both culture systems. B-cell development was
arrested at a stage before expression of the IL-7R . The development
of CD34+CD38 cells into
CD14+ myeloid cells in the S17 system was not inhibited
by overexpression of Id3. Moreover, Id3+ cells, although
inhibited in their B-cell development, were still able to develop into
natural killer (NK) cells when cultured in a combination of Flt-3L,
IL-7, and IL-15. These findings confirm the essential role of bHLH
factors in B-cell development and demonstrate the feasibility of
retrovirus-mediated gene transfer as a tool to genetically modify human
B-cell development.
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