Interest of targeted genome modification techniques in terrestrial livestock species

Des nouvelles nucléases permettent de modifier le génome de nombreuses espèces de façon ciblée ; à un endroit précis et défini dudit génome. Concernant les animaux de rente, les applications envisagées peuvent se classer dans trois grandes perspectives: (i) les projets à visée de recherche fondamentale, lorsque l’espèce murine ne peut pas être utilisée (cornage, saisonnalité, à titre d’exemples) ; (ii) les projets à visée biomédicale pour créer des animaux modèles de pathologies humaines quand le modèle murin n’est pas adéquat ; et (iii) les projets à visée agronomique pour apporter un caractère favorable, décrit dans une autre espèce ou race, à une race ou une espèce ne possédant pas ce caractère. Ces techniques représentent une alternative très efficace à l’introgression, tout en abolissant la barrière d’espèce. Elles pourront également être envisagées en appui à la sélection génomique pour éliminer les allèles défavorables lorsque leur utilisation sera autorisée en Europe, ce qui n’est pas encore le cas.Nowadays, new endonucleases make it possible to target precisely defined alterations in the genomic DNA of many species. With regard to livestock, the envisaged applications can be divided into three broad perspectives: (i) the projects with a basic research objective, when the mouse species cannot be used (horns development, seasonality, as examples) ; (ii) biomedical projects to create animal models of human pathologies when the mouse model is not adequate ; and (iii) agronomic projects aimed at providing an interesting character already described in another species or race, into a race or species not possessing such a character. These techniques represent a very effective alternative to introgression, while abolishing the species barrier. They may also be considered in support of genomic selection to eliminate advers

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

Identification and characterization of new miRNAs cloned from normal mouse mammary gland

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are small non-coding RNAs that have been found to play important roles in silencing target genes and that are involved in the regulation of various normal cellular processes. Until now their implication in the mammary gland biology was suggested by few studies mainly focusing on pathological situations allowing the characterization of miRNAs as markers of breast cancer tumour classes. If in the normal mammary gland, the expression of known miRNAs has been studied in human and mice but the full repertoire of miRNAs expressed in this tissue is not yet available.</p> <p>Results</p> <p>To extend the repertoire of mouse mammary gland expressed miRNAs, we have constructed several libraries of small miRNAs allowing the cloning of 455 sequences. After bioinformatics' analysis, 3 known miRNA (present in miRbase) and 33 new miRNAs were identified. Expression of 24 out of the 33 has been confirmed by RT-PCR. Expression of none of them was found to be mammary specific, despite a tissue-restricted distribution of some of them. No correlation could be established between their expression pattern and evolutionary conservation. Six of them appear to be mouse specific. In several cases, multiple potential precursors of miRNA were present in the genome and we have developed a strategy to determine which of them was able to mature the miRNA.</p> <p>Conclusion</p> <p>The cloning approach has allowed improving the repertoire of miRNAs in the mammary gland, an evolutionary recent organ. This tissue is a good candidate to find tissue-specific miRNAs and to detect miRNA specific to mammals. We provide evidence for 24 new miRNA. If none of them is mammary gland specific, a few of them are not ubiquitously expressed. For the first time 6 mouse specific miRNA have been identified.</p

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

Distal element(s) is(are) required for position-independent expression of the goat α-lactalbumin gene in transgenic mice. Potential relationship with the location of the cyclin T1 locus

We recently reported the site-independent and copy-number-related expression in mice of a goat α-lactalbumin gene with 150 kb and 10 kb of 5'- and 3'-flanking sequences, respectively. In the present study, we observed that the resection of the 5'-flanking region, leaving only 70 kb, resulted in a site-dependent expression of this milk protein-encoding transgene. This suggests that important cis-regulatory elements are located within the distal-deleted sequence. Within this region, we localised the promoter of the cyclin T1 gene, an ubiquitously expressed gene. So far, no other gene has been located between these two loci. Since these two genes are differentially expressed, our data suggest the potential location of an insulator within the deleted region that allows the two genes to be independently regulated

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

Transmission of Atypical Bovine Prions to Mice Transgenic for Human Prion Protein

To assess risk for cattle-to-human transmission of prions that cause uncommon forms of bovine spongiform encephalopathy (BSE), we inoculated mice expressing human PrP Met129 with field isolates. Unlike classical BSE agent, L-type prions appeared to propagate in these mice with no obvious transmission barrier. H-type prions failed to infect the mice