The prion or the related Shadoo protein is required for early mouse embryogenesis

AbstractThe prion protein PrP has a key role in transmissible spongiform encephalopathies but its biological function remains largely unknown. Recently, a related protein, Shadoo, was discovered. Its biological properties and brain distribution partially overlap that of PrP. We report that the Shadoo-encoding gene knockdown in PrP-knockout mouse embryos results in a lethal phenotype, occurring between E8 and E11, not observed on the wild-type genetic background. It reveals that these two proteins play a shared, crucial role in mammalian embryogenesis, explaining the lack of severe phenotype in PrP-knockout mammals, an appreciable step towards deciphering the biological role of this protein family

Substitution of the α-lactalbumin transcription unit by a CAT cDNA within a BAC clone silenced the locus in transgenic mice without affecting the physically linked Cyclin T1 gene

We recently reported that a goat bacterial artificial chromosome (BAC) clone conferred site-independent expression in transgenic mice of the two loci present within its insert, the ubiquitously expressed Cyclin T1 and the mammary specific β-lactalbumin (αlac) genes. To assess if this vector could target mammary-restricted expression of cDNA, the CAT ORF was introduced by homologous recombination in Escherichia coli in place of the αlac transcription unit. The insert of this modified BAC was injected into mice and three transgenic lines were derived. None of these lines expressed the CAT gene suggesting that the use of long genomic inserts is not sufficient to support the expression of intron-less transgenes. The physically linked goat Cyclin T1 locus was found to be active in all three lines. This observation reinforced the hypothesis that the two loci are localised in two separate chromatin domains

Substitution of the ?-lactalbumin transcription unit by a CAT cDNA within a BAC clone silenced the locus in transgenic mice without affecting the physically linked Cyclin T1 gene

We recently reported that a goat bacterial artificial chromosome (BAC) clone conferred site-independent expression in transgenic mice of the two loci present within its insert, the ubiquitously expressed Cyclin T1 and the mammary specific $\alpha$-lactalbumin ($\alpha$lac) genes. To assess if this vector could target mammary-restricted expression of cDNA, the CAT ORF was introduced by homologous recombination in Escherichia coli in place of the $\alpha$lac transcription unit. The insert of this modified BAC was injected into mice and three transgenic lines were derived. None of these lines expressed the CAT gene suggesting that the use of long genomic inserts is not sufficient to support the expression of intron-less transgenes. The physically linked goat Cyclin T1 locus was found to be active in all three lines. This observation reinforced the hypothesis that the two loci are localised in two separate chromatin domains

Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

<p>Abstract</p> <p>Background</p> <p>The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages.</p> <p>Results</p> <p>Only subtle transcriptomic differences resulting from the <it>Prnp </it>knockout could be evidenced, beside <it>Prnp </it>itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal <it>Prnp </it>disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences.</p> <p>Conclusions</p> <p>These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.</p

Submicroscopic Deletions at 13q32.1 Cause Congenital Microcoria.

International audienceCongenital microcoria (MCOR) is a rare autosomal-dominant disorder characterized by inability of the iris to dilate owing to absence of dilator pupillae muscle. So far, a dozen MCOR-affected families have been reported worldwide. By using whole-genome oligonucleotide array CGH, we have identified deletions at 13q32.1 segregating with MCOR in six families originating from France, Japan, and Mexico. Breakpoint sequence analyses showed nonrecurrent deletions in 5/6 families. The deletions varied from 35 kbp to 80 kbp in size, but invariably encompassed or interrupted only two genes: TGDS encoding the TDP-glucose 4,6-dehydratase and GPR180 encoding the G protein-coupled receptor 180, also known as intimal thickness-related receptor (ITR). Unlike TGDS which has no known function in muscle cells, GPR180 is involved in the regulation of smooth muscle cell growth. The identification of a null GPR180 mutation segregating over two generations with iridocorneal angle dysgenesis, which can be regarded as a MCOR endophenotype, is consistent with the view that deletions of this gene, with or without the loss of elements regulating the expression of neighboring genes, are the cause of MCOR

Targeted mutation and inactivation of the kinesin light chain 3 protein-encoding gene have no impact on mouse fertility.

International audienceAbstract The kinesin light chain 3 protein (KLC3) is the only member of the kinesin light chain protein family that was identified in post-meiotic mouse male germ cells. It plays a role in the formation of the sperm midpiece through its association with both spermatid mitochondria and outer dense fibers (ODF). Previous studies showed a significant correlation between its expression level and sperm motility and quantitative semen parameters in humans, while the overexpression of a KLC3-mutant protein unable to bind ODF also affected the same traits in mice. To further assess the role of KLC3 in fertility, we used CRISPR/Cas9 genome editing in mice and investigated the phenotypes induced by the invalidation of the gene or of a functional domain of the protein. Both approaches gave similar results, i.e. no detectable change in male or female fertility. Testis histology, litter size and sperm count were not altered. Apart from the line-dependent alterations of Klc3 mRNA levels, testicular transcriptome analysis did not reveal any other changes in the genes tested. Western analysis supported the absence of KLC3 in the gonads of males homozygous for the inactivating mutation and a strong decrease in expression in males homozygous for the allele lacking one out of the five tetratricopeptide repeats. Overall, these observations raise questions about the supposedly critical role of this kinesin in reproduction, at least in mice where its gene mutation or inactivation did not translate into fertility impairment

Données de base sur la transgenèse

National audienceLa transgenèse permet d’introduire dans le génome d’un animal un fragment d’ADN qui sera ensuite transmis de génération en génération. Elle utilise différentes approches méthodologiques. Certaines, encore limitées à peu d’espèces, permettent des modifications très fines du génome. La transgenèse, associée au développement de la biologie moléculaire, offre des applications multiples tant dans la recherche fondamentale qu’appliquée. Elle est de fait devenue un outil indispensable pour l’analyse de la régulation de l’expression des gènes et la compréhension de leur fonction

Expression de la protéine Doppel dans les cellules de Sertoli : Impact sur la fertilité mâle chez la souris

Expression de la protéine Doppel dans les cellules de Sertoli : Impact sur la fertilité mâle chez la souris. 2. Journées Scientifiques du GdR Repr

Transcriptomic analysis brings new insight into the biological role of the prion protein during mouse embryogenesis

Chantier qualité GAThe biological function of the Prion protein remains largely unknown but recent data revealed its implication in early zebrafish and mammalian embryogenesis. To gain further insight into its biological function, comparative transcriptomic analysis between FVB/N and FVB/N Prnp knockout mice was performed at early embryonic stages. RNAseq analysis revealed the differential expression of 73 and 263 genes at E6.5 and E7.5, respectively. The related metabolic pathways identified in this analysis partially overlap with those described in PrP1 and PrP2 knockdown zebrafish embryos and prion-infected mammalian brains and emphasize a potentially important role for the PrP family genes in early developmental processes