Figure 2: Analysis of Foxp3 transgenic mice.

a, Southern-blot analysis was performed on genomic DNA prepared from tail tips, digested with BstXI (Bs) and probed with the fragment indicated by a heavy horizontal bar in the schematic diagram. At the endogenous Foxp3 locus, this fragment hybridizes to 3.1- and 4.8-kb fragments (shown in schematic, and on Southern blot); in the case of multiple copies of the 30.8-kb HpaI (Hp) transgene integrated in a head-to-tail array, the probe hybridizes to the same internal 3.1-kb fragment as well as an unique 3.6-kb fragment (indicated on Southern blot). Representative samples from normal littermate controls (NLC) and transgene (Tg) lines 1292L, 1292H, 2826, 2827 and 2828 are shown. Using the endogenous 4.8-kb BstXI fragment as an internal control in each lane, transgene copy numbers were determined by analysis with a PhosphorImager 445 SI. The copy numbers indicated in Table 1 are the result of analyzing multiple animals from each transgenic line. Additional higher molecular weight bands most apparent in the analyses of lines 1292H, 2827 and 2828 represent the unique single-copy BstXI fragments generated at the sites of transgene insertion. b, Lymph nodes from NLC, sf/Y mutant, and sf/Y; Tg animals were removed at 12 days of age. Lymphoid organs appeared normal in all sf/Y; Tg animals examined, irrespective of transgene copy number. c, Foxp3 transgene expression level in spleen (S) and thymus (T) roughly correlates with copy number, as determined by northern-blot analysis of total RNA prepared from animals at 10 days of age. A major transcript is seen at ∼2.4 kb and a minor species, at ∼3.7 kb, the difference in length being due to the use of alternative polyadenylation sites. As with endogenous Foxp3 , Tg expression is undetectable in liver (L). We loaded 20 μg total RNA in each lane.