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 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-7Ralpha. 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.
Jaleco
A C
AC
Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Amsterdam, The Netherlands.
Stegmann
A P
AP
Heemskerk
M H
MH
Couwenberg
F
F
Bakker
A Q
AQ
Weijer
K
K
Spits
H
H
D000818:Animals; D017209:Apoptosis; D001402:B-Lymphocytes / Q000166:cytology*; D051792:Basic Helix-Loop-Helix Proteins; D002454:Cell Differentiation / Q000235:genetics; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D018920:Coculture Techniques; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts; D018507:Gene Expression Regulation, Developmental; D005799:Genes, Dominant; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D019409:Interleukin-15 / Q000494:pharmacology; D020381:Interleukin-17 / Q000494:pharmacology; D015851:Interleukin-7 / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000166:cytology; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D009363:Neoplasm Proteins; D020395:Receptors, Interleukin-7 / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D051796:Inhibitor of Differentiation Proteins; D019409:Interleukin-15; D020381:Interleukin-17; D015851:Interleukin-7; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D020395:Receptors, Interleukin-7; D019089:Stem Cell Factor; D014157:Transcription Factors; C084881:flt3 ligand protein; C064658:RAG-1 protein; C075640:ID3 protein, human