Molecular and cellular characterization of the CD41⁺c-Kit^- sorted cells. (A) Expression levels for 4 megakaryocytic genes (GP3a, Gata1, Fog1, Mpl)inwtand transgenic (Tg) CD41⁺c-Kit^--sorted cells were evaluated by real time RT-PCR. The expression level for each gene was normalized with 18S rRNA, and the fold-change ratio was determined by comparing the expression in reconstituted cells to the basal levels found in wt cells. Ethidium bromide-stained gels are shown. (B) CD41⁺c-Kit^- population was purified from wt transgenic mice at D3 and from 5-FU-injected mice that were allowed to recover for 10 days (5FU). Expression levels for 3 lymphoid genes (TdT, Rag2, Ezh2) were compared by real-time RT-PCR. ▪ indicates Tg; ▦, 5FU. Error bars indicate SD. (C) CD41⁺c-Kit^- was allowed to attach to slides coated with polylysine, fixed, permeabilized, and incubated with antibodies against VWF revealed by Cy3-labeled donkey anti-rabbit IgG, anti-CD11b, and anti-B220 revealed by Cy3-streptavidin. Representative fields were digitized using a fluorescence microscope at 63 × magnification. (D) Expression levels of the 230 transcripts found differentially expressed in transgenic compared with wt CD41⁺c-Kit^- (the complete gene list is available in Table S2). These genes were selected using an ANOVA parametric test (Welch t test; P < .05) with Benjamini and Hochberg multiple testing correction. Two clusters were identified as exhibiting similar expression levels in the transgenic populations. Cluster 1 is composed mainly of genes known to be expressed in lymphoid populations. Cluster 2 shows genes that relate the transgenic populations to wt CLP and pro-B populations.