Gene trap screening using negative selection: Identification of two tandem, differentially expressed loci with potential hematopoietic function

A fusion gene between Escherichia coil lacZ and herpes simplex virus thymid ine kinase (HSV-tk) was constructed and used in a gene trap screen for hema topoietic loci in mouse embryonic stem (ES) cells. This gene, galtek, allow ed both convenient histochemical detection of expression as well as ablatio n of expressing cells under 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5- iodouracil (FIAU) selection. individual ES cell clones bearing gene trap in sertions were differentiated in the presence of FIAU and scored for erythro poietic activity at day 9 of differentiation. Screening of a total of 235 i ndependent gene trap lines identified one clone, F3, which consistently dem onstrated FIAU-sensitive erythropoiesis during in vitro differentiation. Cl oning of endogenous transcribed sequences from the F3 insertion site identi fied two distinct transcription units, F3-1 and F3-2, encoding mRNAs of app roximately 1.3 kb and 3.35 kb, respectively. The transcripts were unrelated and did not exhibit similarity to known sequences. Both loci demonstrated similar relative levels of expression in the heart, testis, kidney, and lun g as assessed by Northern blot hybridization. Whole-mount in situ hybridiza tion detected F3-2 expression at multiple sites in embryonic day (E) 10.5 e mbryos, including the genital ridges, the aortic endothelium, and endotheli um-associated cell clusters within the aortic lumen. Expression of F3-2 in the aortic endothelium and endothelium-associated clusters overlapped that of gata-2, a gene required for hematopoietic development. The FIAU sensitiv ity of hematopoiesis in F3 embryoid bodies may result from expression of ga ltek during the formation of early hematopoietic cells, directed by regulat ory signals from one or both of these endogenous loci. (C) 1999 Wiley Liss, Inc.