Ample Pairs

We show that the ample degree of a stable theory with trivial forking is preserved when we consider the corresponding theory of belles paires, if it exists. This result also applies to the theory of $H$-structures of a trivial theory of rank $1$.Comment: Research partially supported by the program MTM2014-59178-P. The second author conducted research with support of the programme ANR-13-BS01-0006 Valcomo. The third author would like to thank the European Research Council grant 33882

Loss of <i>mp29</i> resulted in aberrant embryonic development.

<p>The following results were repeated with three independent experiments. (A) Morphology of embryos cultured <i>in vitro</i>. Embryos were collected from uterus at E3.5 and cultured <i>in vitro</i> to E6.5. Embryos were hatched at E6.5 in <i>mp29^+/+</i> and <i>mp29^GT/+</i> embryos. (B) Embryos were isolated from E7.5 to E9.5 for hematoxylin–eosin staining. <i>mp29^+/+</i> and <i>mp29^GT/+</i> embryos at E7.5 had normal gastrulation. ee: embryonic ectoderm. m: mesoderm. ve: visceral endoderm. (C) Whole embryo extracts at E7.5 were lysed in RIPA buffer for Western blotting analysis. Hsp90 was used as a loading control.</p

<i>mp29</i> transgene complemented the deficiency of mp29.

<p><i>mp29^GT/+</i> mice were mated with <i>mp29^Tg/+</i> mice to generate <i>mp29^GT/+mp29^Tg/+</i> littermates. Inbreed of <i>mp29^GT/+mp29^Tg/+</i> male mice with <i>mp29^GT/+mp29^+/+</i> female mice gave birth to <i>mp29^GT/GTmp29^Tg/+</i> mice. (A) Genotyping of complemented mice by PCR analysis. NSPF1/NeoES PCR products indicated the presence of U3NeoSV1 vector. NSPF1/NSPR1 primers were used to determine whether both of alleles were inserted with U3NeoSV1 vector. mPGKF1/mp29R1 PCR products indicated the presence of <i>mp29</i> transgene with a product of 500 bp. Tg refers to the hemizygous <i>mp29</i> transgene. (B) The genotyping of 76 complemented offsprings was examined. Eight pups exhibited homozygously interrupted genotype but with mp29 transgene (<i>mp29^GT/GTmp29^Tg/+</i>). There were no <i>mp29^GT/GT</i> mice that could survive without the presence of <i>mp29</i> transgene. (C) Tissue extracts of the livers from complemented mice were prepared for Western blot analysis. Note that normal expression levels of α-tubulin, Chk1, and Chk2 were restored in <i>mp29^GT/GTmp29^Tg/+</i> mice.</p

Decreased α-tubulin and Chk1 expression in <i>mp29^GT/GT</i> embryo.

<p>Unless otherwise stated, the following results were repeated with three independent experiments. (A) Whole embryo extracts at E7.5 were prepared for Western blotting analysis. (B) RT-PCR for embryos prepared from E7.5. Note lower levels of α-tubulin, Chk1, and Chk2, but not β-tubulin, in <i>mp29^GT/GT</i> embryos. GAPDH was used as an input control. (C) Total RNAs from embryos at E7.5 were extracted and first strand cDNAs were transcribed for quantitative real-time PCR analysis to determine the relative pre-mRNA and mRNA levels of α-tubulin and Chk1. GAPDH was used as a normalized control. (D) NIH3T3 cells were co-transfected with siRNAs and E1A splicing reporter for <i>in vivo</i> alternative splicing assay. Total RNAs were isolated and subjected to RT-PCR. The splicing products were quantitatively analyzed with three independent experiments. GAPDH was used as a normalized control. * indicated <i>p</i><0.05 in one-way ANOVA F-test.</p

Interruption of mouse <i>mp29</i> gene by a gene trap vector.

<p>(A) Schematic of the targeting vector U3NeoSV1 within exon 2 and 3 of <i>mp29</i> gene. (B) Genotyping of mouse tail DNAs by PCR. NSPF1/NeoES primers were used to examine the presence of U3NeoSV1 vector in <i>mp29</i> gene with a product of 250 bp. NSPF1/R1 primers were used to determine if both of alleles were inserted with U3NeoSV1 vectors with a product of 300 bp. 18s RNA was used as a control. +/+: Wild type mice; GT/+: Heterozygous mice with U3NeoSV1 inserted in one allele. (C) Of 86 offspring mice, 28 pups were identified as wild type (+/+) and 58 pups were heterozygous (GT/+).</p

Analysis of embryos from intercrosses of <i>mp29^GT/+</i> mice.

<p>Analysis of embryos from intercrosses of <i>mp29^GT/+</i> mice.</p

Impaired G2/M checkpoint in <i>mp29^GT/GT</i> embryos.

<p>Unless otherwise stated, the following results were repeated with three independent experiments. (A) <i>mp29^+/+</i>, <i>mp29^GT/+</i>, and <i>mp29^GT/GT</i> blastocysts at E3.5 were treated with aphidicolin (1 µM) for 8 h, incubated with nocodazole (0.1 µg/ml) for an additional 16 h, and then fixed and stained with antibody specific to phosphohistone H3 at Ser10. (B) <i>mp29^+/+</i>, <i>mp29^GT/+</i>, and <i>mp29^GT/GT</i> blastocysts at E3.0 were irradiated with UV light (30 J/m²), incubated with nocodazole for 16 h, and then co-immunostained with anti-phosphohistone H3 at Ser10 antibody and TUNEL fluorescein. The genotypes of each embryo were determined by PCR. Images were obtained using Leica DM6000B microscope.</p