Manipulation and Visualization of the Erythroid Lineage - In Vivo Models for Erythroid Disorders.

Red blood cells have a short half-life and are continuously renewed in a tightly controlled growth process. Dysregulation of erythropoiesis results in erythroleukemias or more frequently in anemias. To elucidate molecular mechanisms regulating red cell production in an animal model, we generated a mouse line (ErGFPcre) that simultaneously facilitates erythroid specific gene manipulation via the CreloxP recombination system and visualization of erythroid progenitor cells. To avoid site of integration effects frequently observed for randomly integrated transgenes, we used a knock-in strategy targeting the genomic EpoR locus that ensures a reliable EpoR promoter controlled transgene expression of our GFPcre fusion protein.

The flow cytometric analysis of GFP fluorescence in different hematopoietic subpopulations of adult and embryonic ErGFPcre mice revealed a strictly erythroid-specific expression pattern for GFPcre. Further studies on GFP positive erythroid progenitor cells indicated a developmental switch in lineage progression from the hematopoietic stem cell compartment to early erythroid progenitor cells that are Sca-1 negative and c-kit high. To monitor previous and persistent GFPcre expression during development and to determine the efficiency of Cre-mediated recombination in our mouse model, we crossed the ErGFPcre mouse line with the LacZ reporter strain R26R. The spatial and temporal analysis of GFPcre-mediated LacZ expression confirmed that within the hematopoietic system GFPcre expression is limited to the erythroid lineage. Surprisingly, non-hematopoietic expression of GFPcre is restricted to the vascular system. It is possible that the observed differential transcriptional activity of the knock-in and the wild-type allele is linked to the absence of intron 2 to 7 in the knock-in locus. The quantitative analysis of the recombination frequency confirmed that GFPcre-mediated recombination is limited to erythroid progenitor cells and showed that it occurs in the adult bone marrow at a frequency of up to 80% and in the fetal liver with an efficiency close to 100%.

Thus, our ErGFPcre mouse model offers the possibility to study regulatory mechanisms in erythroid progenitor cells and facilitates the establishment of mouse models for erythroid disorders.