Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish

<p>Abstract</p> <p>Background</p> <p>Nuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (<it>Carassius auratus</it>), the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation.</p> <p>Results</p> <p>Trout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL) allowed the same hatching rates as the non injected controls (84.8% ± 23). In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage). Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage).</p> <p>Conclusions</p> <p>The best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min after somatic cell injection. Although the experiments were not designed to produce characterized clones, application of these parameters to somatic cell nuclear transfer experiments in enucleated metaphase II oocytes is expected to improve the quality of the reconstructed embryos.</p

Isolation from Cattle of a Prion Strain Distinct from That Causing Bovine Spongiform Encephalopathy

To date, bovine spongiform encephalopathy (BSE) and its human counterpart, variant Creutzfeldt-Jakob disease, have been associated with a single prion strain. This strain is characterised by a unique and remarkably stable biochemical profile of abnormal protease-resistant prion protein (PrP(res)) isolated from brains of affected animals or humans. However, alternate PrP(res) signatures in cattle have recently been discovered through large-scale screening. To test whether these also represent separate prion strains, we inoculated French cattle isolates characterised by a PrP(res) of higher apparent molecular mass—called H-type—into transgenic mice expressing bovine or ovine PrP. All mice developed neurological symptoms and succumbed to these isolates, showing that these represent a novel strain of infectious prions. Importantly, this agent exhibited strain-specific features clearly distinct from that of BSE agent inoculated to the same mice, which were retained on further passage. Moreover, it also differed from all sheep scrapie isolates passaged so far in ovine PrP-expressing mice. Our findings therefore raise the possibility that either various prion strains may exist in cattle, or that the BSE agent has undergone divergent evolution in some animals

Etude fonctionnelle de Pacmmar1, élément transposable de type mariner isolé chez le crabe Pachygrapsus marmoratus

Les éléments transposables mariner sont des séquences d ADN mobiles, qui ont la capacité de changer de position dans le génome. Leur structure est simple : ils contiennent deux séquences répétées et inversées (ITR) encadrant un cadre de lecture ouvert codant la transposase, protéine capable à elle seule de catalyser toutes les étapes de la transposition. Celle-ci s effectue en trois grandes étapes : (1) la fixation spécifique de la transposase sur les ITRs ; (2) le clivage de l élément ; (3) sa réinsertion dans un site cible. Au cours du temps, la plupart des éléments ont perdu leur capacité de déplacement suite à l apparition de mutations au niveau du gène de la transposase. A ce jour, seuls quelques éléments actifs ont été identifiés. Les travaux présentés dans cette thèse portent sur l étude fonctionnelle d un élément mariner isolé chez le crabe Pachygrapsus marmoratus. Deux copies de cet élément ont été isolées. Chacune d elles code une transposase appelée respectivement PacTase1 et PacTase2. L objectif de ces travaux a été de déterminer si ces deux éléments, dont les séquences sont naturellement présentes dans le génome de P. marmoratus, sont actifs ou non. L analyse in-silico des deux transposases a permis d identifier les différentes caractéristiques des éléments mariner actifs. L analyse de leur activité, réalisée in vitro et in vivo en cellules humaine HeLa et en cellules de poisson zèbre Pac2, a permis de mettre en évidence une activité partielle des transposases. Celles-ci se fixent spécifiquement aux ITRs et ont une activité de coupure de l ADN qui semble cependant non spécifique. Ces résultats nous laissent penser que la séquence des transposases possède des mutations par rapport à la séquence de la transposase active, ou que l activité de transposition nécessite la présence de facteurs hôtes qui ne sont pas présents dans les deux types cellulaires étudiés.Mariner transposable elements are mobile DNA sequences which are able to move in genomes. Their structure is simple: they contain two inverted terminal repeats (ITR) on each ends of an open reading frame encoding a transposase. Transposition involves three major steps: (1) specific binding of the transposase to ITRs ; (2) cleavage of the element (3) reinsertion into a target site. Most elements lost their ability to move because of mutations in the transposase gene over times. So far, only a few elements were identified to be active. This work focused on the study of the functionality of a mariner element isolated from the crab Pachygrapsus marmoratus. Two copies of this element were isolated which encodes transposase named PacTase1 and PacTase2 respectively. The aim of this work was to determine whether these elements whose sequences are naturally present in the genome of P. marmoratus, are active or not. In-silico analyses show that the two identified transposases display the characteristics of active mariner elements. In vitro and in vivo analyses of their activity, conducted in human HeLa cells and zebrafish Pac2 cells reveal a partial transposition activity. They bind specifically to ITRs and cut DNA. However, cleavage seems to be non specific. These results suggest that the sequence of transposases present mutations compared to the sequence of the active transposase, or that the transposition activity requires the presence of host factors that are not present in these two cell types.LE MANS-BU Sciences (721812109) / SudocSudocFranceF

Import nucléaire de facteurs ovocytaires dans les cellules somatiques en culture : une avancée prometteuse pour leur reprogrammation en vue du transfert nucléaire

Import nucléaire de facteurs ovocytaires dans les cellules somatiques en culture : une avancée prometteuse pour leur reprogrammation en vue du transfert nucléaire. Journées d'Animation Scientifique du département Phase (JAS Phase 2018

Subculture of fin explants: a powerful optimization of fin cell production for cryobanking purposes

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18 Characterisation of early embryonic cellular defects after somatic cell nuclear transfer in fish

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DNA Binding Specificity and Cleavage Activity of Pacmmar TransposaseRID C-6465-2009

International audienceMariner-like elements (MLEs) are members of the Tc1/mariner superfamily of transposable elements which transpose by a "cut and paste" mechanism. Most of the MLEs characterized to date are transpositionally inactive due to the accumulation of mutations in their transposase gene. Here, we report the biochemical study of two copies of the Pacmmar element (Pacmmar1.1 and Pacmmar1.2), isolated from the coastal crab Pachygrapsus marmoratus. These two copies present an open reading frame encoding a putative active transposase. Using an in vitro transposition assay, we show that Pacmmar transposases are unable to perform by themselves the transposition reaction. However, we demonstrate by an electrophoretic mobility shift assay that both transposases bind specifically to the inverted terminal repeat of the Pacmmar element. Moreover, an in vitro cleavage assay showed that both transposases have the capacity to cleave the transposon. The in vitro cleavage activity of Pacmmar transposases appears imprecise, suggesting the requirement of specific host factors or the presence of mutations which have modified the cleavage specificity of the enzyme

Somatic cell nuclear transfer in non-enucleated goldfish oocytes: understanding DNA fate during oocyte activation and first cellular division

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid ofspring which bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fsh, because enucleation is difcult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explores which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fsh oocyte enucleation. The fate of the somatic and maternal DNA during oocyte activation and frst cell cycle was studied using DNA labeling and immunofuorescence in goldfsh clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally afected after oocyte activation. During the frst cell cycle, only 40% of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80% of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed