In:
Blood, American Society of Hematology, Vol. 94, No. 8 ( 1999-10-15), p. 2637-2646
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
1528-0020
,
0006-4971
DOI:
10.1182/blood.V94.8.2637.420k22_2637_2646
Language:
English
Publisher:
American Society of Hematology
Publication Date:
1999
detail.hit.zdb_id:
1468538-3
detail.hit.zdb_id:
80069-7
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