Persistent Expression of the Embryonic Globin, epsilony, in Sox6-Deficient Mice
The epsilony globin gene was initially identified as an upregulated transcript in the p100H mouse using subtractive hybridization to identify Sox6 downstream targets. This initial observation was confirmed in an independent knock-out allele of Sox6 [13] using real-time polymerase chain reaction (PCR) (unpublished data). Real-time PCR was used to quantitate the expression levels of other globin genes in p100H mutant and WT mouse livers at two developmental stages, 15.5 dpc and 18.5 dpc. As shown in Figure 1, the epsilony gene is expressed at high levels at both time points in mutant mice, in contrast to the decline in expression observed in WT mice. No difference was seen between WT and heterozygous mice (unpublished data). Interestingly, the expression levels of the other two embryonic globin genes (zeta and betah1) are also higher in p100H homozygous mice, compared with WT mice, but to a much lesser extent than seen for epsilony globin at 18.5 dpc (Figure 1). Moreover, the expression level of adult beta globin is also somewhat higher in p100H homozygous mice than in WT mice at 18.5 dpc (Figure 1). Perinatal lethality of mutant mice (presumably from the heart defect [14]) precludes us from evaluating postnatal globin expression. The graphs in Figure 1 illustrate real time PCR results that were performed in triplicate (standard deviation of the data is shown by error bars). Because all of the assays were performed at the same time with the same internal control, the levels shown are relative levels and are thus comparable across all samples and are in agreement with previously published results for WT fetal mice [29]. We note that the level of epsilony expression in the livers of 15.5 dpc and 18.5 dpc homozygous mutant mice is statistically equivalent to the level of betamaj/min expression in the livers of 15.5 dpc and 18.5 dpc homozygous WT mice. 
