SummaryNot much is known about the molecular and functional features of pluripotent stem cells (PSCs) in rabbits. To address this, we derived and characterized 2 types of rabbit PSCs from the same breed of New Zealand White rabbits: 4 lines of embryonic stem cells (rbESCs), and 3 lines of induced PSCs (rbiPSCs) that were obtained by reprogramming adult skin fibroblasts. All cell lines required fibroblast growth factor 2 for their growth and proliferation. All rbESC lines showed molecular and functional properties typically associated with primed pluripotency. The cell cycle of rbESCs had a prolonged G1 phase and a DNA damage checkpoint before entry into the S phase, which are the 2 features typically associated with the somatic cell cycle. In contrast, the rbiPSC lines exhibited some characteristics of naïve pluripotency, including resistance to single-cell dissociation by trypsin, robust activity of the distal enhancer of the mouse Oct4 gene, and expression of naïve pluripotency-specific genes, as defined in rodents. According to gene expression profiles, rbiPSCs were closer to the rabbit inner cell mass (ICM) than rbESCs. Furthermore, rbiPSCs were capable of colonizing the ICM after aggregation with morulas. Therefore, we propose that rbiPSCs self-renew in an intermediate state between naïve and primed pluripotency, which represents a key step toward the generation of bona fide naïve PSC lines in rabbits.
-As 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. chicken / radiation hybrid / mapping
-A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q−;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.
Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH -null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.
-Eight new cases of reciprocal translocation in the domestic pig are described. All the rearrangements were highlighted using GTG banding techniques. Chromosome painting experiments were also carried out to confirm the proposed hypotheses and to accurately locate the breakpoints. Three translocations, rcp(4;6)(q21;p14), rcp(2;6)(p17;q27) and rcp(5;17)(p12;q13) were found in boars siring small litters (8.3 and 7.4 piglets born alive per litter, on average, for translocations 2/6 and 5/17, respectively). The remaining five, rcp(5;8)(p12;q21), rcp(15;17)(q24;q21), rcp(7;8)(q24;p21), rcp(5;8)(p11;p23) and rcp(3;15)(q27;q13) were identified in young boars controlled before entering reproduction. A decrease in prolificacy of 22% was estimated for the 3/15 translocation after reproduction of the boar carrier. A parental origin by inheritance of the translocation was established for the (5;8)(p11;p23) translocation.
cDNA clones from a pig granulosa cell cDNA library were isolated by differential hybridisation for follicle stimulating hormone (FSH) regulation in granulosa cells in a previous study. The clones that did not match any known sequence were studied for their expression in granulosa cells (treated or not by FSH) and in fresh isolated ovarian follicles mainly by comparative RT-PCR analysis. These results give functional data on genes that may be implicated in follicular growing. These ESTs have been localised on the porcine genome, using a somatic cell hybrid panel, providing new type I markers on the porcine map and information on the comparative map between humans and pigs.
Les cartes géniques des espèces domestiques sont restées très rudimentaires, voire inexistantes, jusqu’au début des années 1990. La situation a évolué à cette période avec la mise en place de vastes programmes de cartographie du génome, aussi bien au plan national (programmes de cartographie de l’INRA, programmes Génome Français) qu’européen (programmes Bridge) pour quelques espèces majeures dont les espèces bovine et porcine. Il est en effet apparu clairement que la connaissance du génome, indispensable pour l’isolement et le clonage des gènes d’intérêt agronomique, passait par la construction des cartes génétique (mise en place d’un réseau de marqueurs polymorphes par analyse de liaison sur des familles de référence) et cytogénétique (localisation de marqueurs et de gènes sur les chromosomes). Les principales méthodes mises au point pour établir, dans un premier temps, la carte cytogénétique puis, dans un deuxième temps, la carte physique à haute résolution sont présentées.
Les cartes porcines sont maintenant bien développées : près de 1200 microsatellites sont cartographiés sur les familles de référence et 2000 nouveaux sont en cours de développement ; environ 400 gènes ou EST (expressed sequence tags) et 400 marqueurs génétiques sont positionnés sur les cartes cytogénétiques ; les cartes d’irradiation sont en plein essor ; près de 1000 marqueurs et gènes sont ordonnés avec une résolution 10 à 20 fois supérieure à la carte génétique. L’estimation de la taille physique du génome du porc est de 2700 Mb organisées en 38 (2x18 + XY) chromosomes, les cartes génétiques couvrent 2300 cM, et les cartes d’irradiation de 30 à 40000 cR7000. La réalisation d’un contig de BAC global du génome et l’établissement d’un catalogue des gènes sont les deux prochains développements importants de la génomique porcine.
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