Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3

Transgenic and gene targeted mice have contributed greatly to our understan ding of the mechanisms underlying B-cell development. We describe here a mo del system that allows us to apply molecular genetic techniques to the anal ysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent prote in by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver ce lls, cultured overnight in a combination of stem cell factor and interleuki n-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene en coding the dominant negative helix loop helix (HLH) factor Id3 that inhibit s many enhancing basic HLH transcription factors into this vector. CD34(+)C D38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the mur ine 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 developme nt of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7R alpha. 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) ce lls when cultured in a combination of Flt-3L, IL-7, and IL-15. These findin gs confirm the essential role of bHLH factors in B-cell development and dem onstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development. (C) 1999 by The American Socie ty of Hematology.