Véronique Blanquet

The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows: (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.

Phex^L222P mouse is a new ENU mouse model for XLH disease due to Leu to Pro amino acid modification at position 222. Phex^L222P mouse is characterized by growth retardation, hypophosphatemia, hypocalcemia, reduced body bone length, and increased epiphyseal growth plate thickness and femur diameter despite the increase in PHEX^L222P expression. Actually, Phex^L222P mice show an increase in Fgf23, Dmp1, and Mepe and Slc34a1 (Na-Pi IIa cotransporter) mRNA expression similar to those observed in Hyp mice. Femoral osteocalcin and sclerostin and Slc34a1 do not show any significant variation in Phex^L222P mice. Molecular dynamics simulations support the experimental data. P222 might locally break the E217-Q224 β-sheet, which in turn might disrupt inter-β-sheet interactions. We can thus expect local protein misfolding, which might be responsible for the experimentally observed PHEX^L222P loss of function. This model could be a valuable addition to the existing XLH model for further comprehension of the disease occurrence and testing of new therapies.

Nous présentons une nouvelle stratégie instrumentale et computationnelle appelée FAMOUS (pour fast algorithm for three-dimensional (3D) multiphoton microscopy of biomedical structures) basée sur une approche de microscopie multiphotonique assistée par calcul. Le but est l’amélioration visuelle des images d'échantillons biologiques épais offrant ainsi un nouveau point de vue sur les structures biologiques. L'approche de post-traitement repose sur un algorithme de restauration d'image régularisé, alimenté par une estimation 3D précise de la fonction d'étalement du point (Point Spread Function en anglais, PSF) de l'instrument sur toute la profondeur des structures. Cette dernière étape revient à mesurer, grâce à un algorithme d'ajustement de modèle avancé, les distorsions variant en profondeur de l'image résultant de la combinaison entre la contribution instrumentale et les hétérogénéités du milieu. Les performances du pipeline FAMOUS sont évaluées pour un milieu hétérogène constitué d’un muscle entier de souris. La génération de seconde harmonique (SHG), émise par l'assemblage des chaines de myosine du muscle est enregistrée. Les artefacts optiques issus de la chaîne d'acquisition incluant des hétérogénéités dans les 3 dimensions sont estimés avec les spécificités propres à l’échantillon puis retirées numériquement. Des images brutes et restaurées sur 5 µm de l’ultrastructure fine du muscle illustrent la robustesse du pipeline FAMOUS.

BISCEm (https://www.unilim.fr/biscem/), acronyme de Biologie Intégrative Santé Chimie Environnement, est une plateforme technologique créée par l’Institut de recherche GEIST en 2017. La plateforme regroupe des équipements de pointe permettant la réalisation d’analyses moléculaires, cellulaires et tissulaires et d’expériences sur des animaux, dans le strict respect de la règlementation en vigueur et du bien-être animal. La réalisation des prestations, la formation à l’utilisation des équipements, les conseils dans la conception des expériences et dans l’interprétation des résutats, la veille technologique et la gestion de la plateforme sont assurés par une équipe d’une vingtaine d’agents hautement qualifiés. La plateforme est ouverte à toutes les structures de recherche, publiques ou privées.

Background: Linkage disequilibrium (LD) is a key parameter to study the history of populations and to identify and fine map quantitative trait loci (QTL) and it has been studied for many years in animal populations. The advent of new genotyping technologies has allowed whole-genome LD studies in most cattle populations. However, to date, long-range LD (LRLD) between distant variants on the genome has not been investigated in detail in cattle. Here, we present the first comprehensive study of LRLD in French beef cattle by analysing data on 672 Charolais (CHA), 462 Limousine (LIM) and 326 Blonde d’Aquitaine (BLA) individuals that were genotyped on the Illumina BovineHD Beadchip. Furthermore, whole-genome LD and haplotype block structure were analysed in these three breeds. Results: We computed linkage disequilibrium ( r ² ) values for 5.9, 5.6 and 6.0 billion pairs of SNPs on the 29 autosomes of CHA, LIM and BLA, respectively. Mean r ² values drop to less than 0.1 for distances between SNPs greater than 120 kb. However, for the first time, we detected the existence of LRLD in the three main French beef breeds. In total, 598, 266, and 795 LRLD events ( r ² ≥ 0.6) were detected in CHA, LIM and BLA, respectively. Each breed had predominantly population-specific LRLD interactions, although shared LRLD events occurred in a number of regions (55 LRLD events were shared between two breeds and nine between the three breeds). Examples of possible functional gene interactions and QTL co-location were observed with some of these LRLD events, which suggests epistatic selection. Conclusions: We identified long-range linkage disequilibrium for the first time in French beef cattle populations. Epistatic selection may be the main source of the observed LRLD events, but other forces may also be involved. LRLD information should be accounted for in genome-wide association studies.

Muscle atrophy is associated with many diseases including genetic disorders, sarcopenia, or cachexia syndromes. Myostatin ( Mstn ), a transforming growth factor‐beta (TGF‐β) member, plays a key role in skeletal muscle homeostasis as a powerful negative regulator. Over the last decade, about 15 clinical trials aimed at inhibiting the Mstn pathway, failed to produce conclusive results. In this context, we investigated whether growth and differentiation factor‐associated serum protein‐1 (GASP‐1) or GASP‐2, two natural inhibitors of Mstn , might represent a potential therapeutic. As we previously reported, mice overexpressing Gasp‐1 ( Tg(Gasp‐1) ) present an increase of muscle mass but develop metabolic disorders with aging. Here, we showed that overexpression of Gasp‐2 increases the muscular mass without metabolic defects. We also found that Tg(Gasp‐2) mice displayed, like Mstn ^–/– mice, a switch from slow‐ to fast‐twitch myofibers whereas Tg(Gasp‐1) mice exhibit a reverse switch. Our studies supported the fact that GASP‐2 has less affinity than GASP ‐ 1 for Mstn , leading to a constitutive Mstn upregulation only in Tg(Gasp‐1) mice, responsible for the observed phenotypic differences. Altogether, our findings highlighted a gene expression regulatory network of TGF‐β members and their inhibitors in muscle.

Knowledge of population structure is essential to improve the management and conservation of farm animal genetic resources. Microsatellites, which have long been popular for this type of analysis, are more and more neglected in favor of whole-genome single nucleotide polymorphism (SNP) chips that are now available for the main farmed animal species. In this study, we compared genetic patterns derived from microsatellites to that inferred by SNPs, considering three pairs of datasets of sheep and cattle. Population genetic differentiation analyses (Fixation index, F_ST), as well as STRUCTURE analyses showed a very strong consistency between the two types of markers. Microsatellites gave pictures that were largely concordant with SNPs, although less accurate. The best concordance was found in the most complex dataset, which included 17 French sheep breeds (with a Pearson correlation coefficient of 0.95 considering the 136 values of pairwise F_ST, obtained with both types of markers). The use of microsatellites reduces the cost and the related analyses do not require specific computer equipment (i.e., information technology (IT) infrastructure able to provide adequate computing and storage capacity). Therefore, this tool may still be a very appropriate solution to evaluate, in a first stage, the general state of livestock at national scales. At a time when local breeds are disappearing at an alarming rate, it is urgent to improve our knowledge of them, in particular by promoting tools accessible to the greatest number.

While GASP‐1 and GASP‐2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the non‐competitive and competitive anti‐trypsin activities of the full‐length GASP‐1 and GASP‐2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the anti‐trypsin activity of GASP‐1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP‐1 and GASP‐2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP‐2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly‐interacting glutamine residue.