Journal: The Journal of Experimental Medicine
Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs
Figure Lengend Snippet: Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , Smug1 , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.
Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).
Techniques: Mutagenesis, Isolation, Mouse Assay, Injection, Two Tailed Test, MANN-WHITNEY, shRNA, Transduction, Inhibition