3D organization of telomeres in porcine neutrophils and analysis of LPS-activation effect

Background: While the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.[br/] Results: The positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.[br/] Conclusions: The percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that “chromosome bending” occurs in neutrophils as previously observed in gametes

Evolutionary breakpoints through a high-resolution comparative map between porcine chromosomes 2 and 16 and human chromosomes

AbstractThis study reports a high-resolution comparative map between human chromosomes and porcine chromosomes 2 (SSC2) and 16 (SSC16), pointing out new homologies and evolutionary breakpoints. SSC2 is of particular interest because of the presence of several important QTLs. Among 226 porcine ESTs selected according to their expected localization, 151 were RH mapped and ordered on SSC2. This study confirmed the extensive conservation between SSC2 and HSA11 and HSA19 and refined the homology with HSA5 (three blocks defined). Furthermore the SSC2q pericentromeric region was shown to be homologous to another human chromosome (HSA1). A complex organization of these syntenies was demonstrated on SSC2q. Our strategy led us to improve also the SSC16 RH map by adding 45 markers. Two-color fluorescence in situ hybridization of markers representative of each synteny confirmed block order. Finally, 29 breakpoints were identified in both species, and porcine BACs containing two breakpoints were isolated

330 Derivation of porcine induced pluripotent stem cells from fibroblasts of a translocated azoospermic boar

International audienceChromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species

Sedimentary and geomorphic evidence of Saharan megalakes: a synthesis

International audienceAs a first step towards the development of radiation hybrid maps, we have produced a radiation hybrid panel in the chicken by fusing female embryonic diploid fibroblasts irradiated at 6 000 rads with HPRT-deficient hamster Wg3hCl$_2$ cells. Due to the low retention frequency of the chicken fragments, a high number of clones was produced from which the best ones were selected. Thus, 452 fusion clones were tested for retention frequencies with a panel of 46 markers. Based on these results, 103 clones with a mean marker retention of 23.8% were selected for large scale culture to produce DNA in sufficient quantities for the genotyping of numerous markers. Retention frequency was tested again with the same 46 markers and the 90 best clones, with a final mean retention frequency of 21.9%, were selected for the final panel. This panel will be a valuable resource for fine mapping of markers and genes in the chicken, and will also help in building BAC contigs

Non integrative strategy decreases chromosome instability and improves endogenous pluripotency genes reactivation in porcine induced pluripotent-like stem cells

International audienceThe pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and nonintegrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians