miR-144/451eGFP is a quantitative marker of erythroid lineage output.MiR-144/451+/eGFP cells can give rise to (A) G/M cells, (B) B and T cells, and (C) erythroid cells, and marks all erythrocytes in the peripheral blood of bone marrow–transplanted lethally irradiated Ly5.1/5.2 recipients. (i) Representative FACS plots of peripheral blood cells stained for (A) Gr-1 and Mac-1, (B) B220 and CD5, and (C) Ter119. (A-B) Representative FACS plots show the gated populations stained for Ly5.1 and Ly5.2 isolated from animals reconstituted with (ii) Ly5.1 or (iii) miR-144/451+/eGFP:Ly5.2 cells, respectively. Note that a minor population (∼ 15%) of radioresistant recipient-derived T cells can be detected in both wild-type and miR-144/451+/eGFP injected animals. (C) Bottom histograms show gated RBCs plotted for GFP expression (x-axis) from animals reconstituted with (ii) Ly5.1 or (iii) miR-144/451+/eGFP:Ly5.2 cells, respectively. (A-C) Numbers indicate mean fraction of the various gated cell populations based on 5 independent measurements. (D) Experimental setup to evaluate miR-144/451eGFP expression as a quantitative erythroid lineage marker. Lethally irradiated Ly5.1/5.2 recipients were injected with 2 million nucleated whole bone marrow cells representing diverse mixtures of Ly5.1 or miR-144/451+/eGFP:Ly5.2 cells. Eight weeks after transplantation, the fraction of GFP-positive RBCs and Ly5.2 single-positive leukocytes was measured in the peripheral blood. (E) Near-perfect linear correlation between Ly5 chimerism and GFP-positive RBCs. Plot indicates fractions of GFP-positive erythrocytes (x-axis) and Ly5.2 single-positive leukocytes (y-axis) for individual transplanted mice as in panel D. Empty and filled symbols represent individual animals in 4 independent transplantation experiments. The solid line represents simple linear regression based on data points from all experiments. The slope, standard error, and r squared (r2) value computed for each individual experiment as well as for all data points are listed.
