Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice

Abstract

Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .

Developing HSCs become Notch independent by the end of maturation in the AGM region

“…Wild-type and transgenic mouse lines (all C57BL/6, CD45.2/2) used were: (1) a pHes1-d2EGFP reporter of Hes1 expression, 55 (2) Rosa CreERT2 (from L. Grotewold and A. Smith, Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge, UK), (3) sGFP where green fluorescent protein (GFP) is expressed upon Cre-mediated activation, 56 and (4) floxed RBP-Jk. 37 The following primers were used for genotyping by polymerase chain reaction (PCR): (1) …”

“…37,56 Acute RBP-Jk ablation was induced in E10.5 AGM cell suspension using 2-hour incubation with 4-OHT and after reaggregation and 5 days of culture, the generation of dHSCs and CFU-C was assessed functionally (Figure 4E-F). Concurrent Cre-mediated activation of GFP expression upon tamoxifen induction was used as a surrogate marker for Cre-mediated deletion of RBP-Jk and for tracing donor-derived hematopoietic contribution upon transplantation (supplemental Figure 5A).…”

Pure populations of murine macrophages from cultured embryonic stem cells. Application to studies of chemotaxis and apoptotic cell clearance

“…For some experiments the ESC line, GFP#7a, which expresses enhanced green fluorescent protein (eGFP) constitutively (Gilchrist et al, 2003), was used. Both lines behaved similarly in the macrophage differentiation protocols described.…”

Induction of Hematopoietic Differentiation of Mouse Embryonic Stem Cells by an AGM-Derived Stromal Cell Line is Not Further Enhanced by Overexpression of HOXB4

“…CGR8 ES cells, 7a-GFP ES cells [ 45 ], Bry-201 ES cells [ 46 ], and HOXB4-ER T2 hormone inducible ES cell clones (Jackson et al, manuscript in preparation) were maintained in their undifferentiated state as described previously [ 43 ].…”