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We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated sig-

Using an expression gene trapping strategy, we have identified and characterized two novel hematopoietic genes, Hzf and Hhl. Embryonic stem (ES) cells containing a gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to generate mice containing the gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C(2)H(2)-type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression trapping to identify novel hematopoietic genes. Future studies of Hzf and Hhl should provide valuable information on the role these genes play during megakaryocytopoiesis.

An adherent cell line was established from the much studied pre-B cell line 70Z/3. The adherent cell line had the morphological features of a macrophage and stained positive for non-specific esterase. In addition, this line expressed high levels of RNA for the macrophage specific enzyme, lysozyme. Although the 70Z/3 macrophage variant has lost the ability to respond to lipopolysaccharide and interferon-gamma by expressing RNA transcripts for immunoglobulin light chain, it exhibited a new response characterized by increased levels of I-A RNA transcripts. Both the pre-B and macrophage variants also expressed RNA transcripts which hybridize to a Hox 2.3 probe. In contrast, transcripts which hybridize to Hox 1.1, 2.1, and 6.1 probes are expressed in the pre-B line but could not be detected in the macrophage.

In a retinoic acid (RA) gene trap screen of mouse embryonic stem (ES) cells, a novel gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to determine the expression pattern of the gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryogenesis, Aqr is expressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expression was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The gene-trapped Aqr locus was transmitted through the mouse germ line in three genetic backgrounds. In the F2 generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The gene trap insertion, however, does not seem to generate a null mutation, because Aqr transcripts are still present in the homozygous mutant animals. The Aqr open reading frame has weak homology to RNA-dependent RNA polymerases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis.

In a retinoic acid (RA) gene‐trap screen of mouse embryonic stem (ES) cells, a novel gene, named Aquarius (Aqr), was identified and characterized. The promoterless lacZ marker was used to trap the genomic locus and to determine the expression pattern of the gene. Aqr transcripts are strongly induced in response to RA in vitro. During embryogenesis, Aqr is expressed in mesoderm, in the neural crest and its target tissues, and in neuroepithelium. Expression was first detected at 8.5 days postcoitum, when neural crest cells are visible at the lateral ridges of the neural plate. The gene‐trapped Aqr locus was transmitted through the mouse germ line in three genetic backgrounds. In the F2 generation, the expected mendelian ratio of 1:2:1 was observed in all backgrounds, indicating that homozygous mice are viable. Homozygotes are normal in size and weight and breed normally. The gene trap insertion, however, does not seem to generate a null mutation, because Aqr transcripts are still present in the homozygous mutant animals. The Aqr open reading frame has weak homology to RNA‐dependent RNA polymerases (RRPs) of the murine hepatitis viruses and contains an RRP motif. Aqr was mapped to mouse chromosome 2 between regions E5 through F2 by using fluorescence in situ hybridization analysis. Dev. Dyn. 1998;212:304–317. © 1998 Wiley‐Liss, Inc.

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