Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR - PubMed (original) (raw)

Figure 2

Analysis of transgene expression. (A) Western blot analysis of Myc–HuR expression using the 9E10 anti-Myc antibody on samples from 293 cells (either untransfected (lane 1), or transfected with the β-actin–HuR (BA–HuR) construct (lane 2), and from various organs, as indicated, from wild-type (lanes 3, 6, 10, 13, 18 and 21), BA–HuR line 18 (lanes 4, 5, 7, 8, 11, 15, 17, 20 and 23) or BA–HuR line 6 (lanes 9, 12, 14, 16, 19 and 22) mice. (B) Comparison of the methylation status of β-actin regulatory sequences in BA–HuR and β-actin–Auf1 mice. A Southern blot was carried out on DNA that was extracted from brain, digested with the indicated restriction enzymes, and hybridized with a β-actin probe. The structure of the β-actin–HuR transgene and the location of the _Msp_I and _Hpa_II sites are shown in the bottom panel. (C) Western blot analysis of transgene expression in BA–GH line 21 using anti-green-fluorescent-protein (anti-GFP) and anti-Myc antibodies to analyse samples from spleen, testis, brain, thymus and heart. C+ indicates a C1 mouse from the BA–HGdel line 21 different to that used for the first five lanes. (D). Myc–HuR (transgenic) and endogenous HuR expression, as detected by using the anti-Myc or anti-HuR antibodies on testes from BA–HGdel lines 21, 27 and 31 (from the C1 and C2 generations) and from wild-type mice. (E) Northern blot analysis was carried out on RNA extracted from muscle, lung and testis from BA–HGdel line 21 and line 27 mice, and the blot was hybridized with an HuR or GAPDH probe. BA–Auf1, β-actin–Auf1; SV40, simian virus 40; WT, wild type.