Figure 5.
Figure 5. TCRβ locus rearrangement does not require the presence of Gli3. (A) Southern blot hybridizations were performed on PCR products amplified using primers specific for TCRDβ2 and TCRJβ2.7 and DNA templates from DN subsets sorted from E15.5 thymus. The probe used corresponded to a germ line (unrearranged) TCRβ fragment and was obtained by radio-labeling a PCR product amplified from genomic DNA using the TCRDβ2 and TCRJβ2.7 specific oligos. PCR was performed on DN1, DN2, DN3, and DN4 thymocyte populations sorted from wt, Gli3+/–, and Gli3–/– mice. The PCR reactions were stopped when all were still in the exponential phase as determined by the titration of templates. (B) The extent of TCRβ rearrangement observed in panel A was quantified by measuring the total intensity of the 6 rearranged bands and relating back to the intensity of the germ line band (uppermost band in each panel) by dividing the total intensity of the bands corresponding to the rearranged locus by the intensity of the band corresponding to nonrearranged locus. The levels of rearrangement observed in the wt mice (▪) was set to 1 and the levels of rearrangement observed in Gli3+/– (▦) and Gli3–/– mice (□) expressed relative to this level. No reduction in TCRDβ2-Jβ2.7 rearrangement was observed in DN3 and DN4 thymic subsets isolated from Gli3+/– or Gli3–/– mice. (C) TCRβ V-(D)J rearrangements were analyzed using the same technique as in panel A, except that the PCR products were generated using 5′ primers specific to TCR Vβ5.1 (left) and TCR Vβ8.2. (right) together with the same 3′ primer to Jβ2.7 as in panel A. In addition the genomic DNA content for each extraction was calculated by real-time PCR, and the same starting template for the rearrangement PCRs was used in all reactions. The probe used was the same as in panel A. TCRβ V(D)J rearrangement had occurred in DN3 and DN4 thymic subsets isolated from wt, Gli3+/–, and Gli3–/– mice. The PCR reactions were stopped when all were still in the exponential phase as determined by the titration of templates. (D) The levels of rearrangement observed in panel C was quantified by using equivalent starting amounts of genomic DNA as determined by real-time PCR and measuring the total intensity of all the bands and relating this amount back to that observed in DNA isolated from the wt mouse. No significant differences were observed in the level of TCRβ V(D)J rearrangement observed in wt (▪), Gli3+/– (▦), and Gli3–/– (□) mice.

TCRβ locus rearrangement does not require the presence of Gli3. (A) Southern blot hybridizations were performed on PCR products amplified using primers specific for TCRDβ2 and TCRJβ2.7 and DNA templates from DN subsets sorted from E15.5 thymus. The probe used corresponded to a germ line (unrearranged) TCRβ fragment and was obtained by radio-labeling a PCR product amplified from genomic DNA using the TCRDβ2 and TCRJβ2.7 specific oligos. PCR was performed on DN1, DN2, DN3, and DN4 thymocyte populations sorted from wt, Gli3+/–, and Gli3–/– mice. The PCR reactions were stopped when all were still in the exponential phase as determined by the titration of templates. (B) The extent of TCRβ rearrangement observed in panel A was quantified by measuring the total intensity of the 6 rearranged bands and relating back to the intensity of the germ line band (uppermost band in each panel) by dividing the total intensity of the bands corresponding to the rearranged locus by the intensity of the band corresponding to nonrearranged locus. The levels of rearrangement observed in the wt mice (▪) was set to 1 and the levels of rearrangement observed in Gli3+/– (▦) and Gli3–/– mice (□) expressed relative to this level. No reduction in TCRDβ2-Jβ2.7 rearrangement was observed in DN3 and DN4 thymic subsets isolated from Gli3+/– or Gli3–/– mice. (C) TCRβ V-(D)J rearrangements were analyzed using the same technique as in panel A, except that the PCR products were generated using 5′ primers specific to TCR Vβ5.1 (left) and TCR Vβ8.2. (right) together with the same 3′ primer to Jβ2.7 as in panel A. In addition the genomic DNA content for each extraction was calculated by real-time PCR, and the same starting template for the rearrangement PCRs was used in all reactions. The probe used was the same as in panel A. TCRβ V(D)J rearrangement had occurred in DN3 and DN4 thymic subsets isolated from wt, Gli3+/–, and Gli3–/– mice. The PCR reactions were stopped when all were still in the exponential phase as determined by the titration of templates. (D) The levels of rearrangement observed in panel C was quantified by using equivalent starting amounts of genomic DNA as determined by real-time PCR and measuring the total intensity of all the bands and relating this amount back to that observed in DNA isolated from the wt mouse. No significant differences were observed in the level of TCRβ V(D)J rearrangement observed in wt (▪), Gli3+/– (▦), and Gli3–/– (□) mice.

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