New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreERT2 transgene at the otx2 gene locus to generate a conditional Cre-driver line.We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreERT2 upstream of the endogenous ATG of otx2, we utilized this gene’s native promoter and enhancer elements to perfectly match CreERT2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreERT2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

No description available.

No description available.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The aim of this project was to produce and study a murine homozygous knock-in model containing a fast skeletal regulatory light chain (RLC) containing a Asp49toAla point mutation. The D49A mutation is in the functional calcium binding loop of RLC, which is believed to modulate muscle contraction in striated muscle. To introduce the mutation, a reversible knock-out/knock-in system was employed. The Cre/Lox-P strategy was used to conditionally knock-in the RLC D49A mutation. The generation of the knock-in mouse was attempted with two different breeding strategies consisting of two Cre mouse lines with differential expression patterns during development. The proposed animal was never produced because the RLC knock-out recombination event introduced a splicing error resulting in a stop codon in intron 2. Extensive DNA, RNA and protein analysis as well as histological, gross morphology and muscle physiology studies obtained from the animals of the two breeding strategies lead to the identification of the splicing error. Evidence for this outcome is presented. A recommendation for a different strategy in future studies is included.

Η geminin είναι ένα σχετικά καινούριο μόριο το οποίο διαθέτει καίριο ρόλο κατά την ανάπτυξη και την διαφοροποίηση, εξαιτίας του νευροποιητικού δυναμικού της, της ικανότητάς της να αλληλεπιδρά με μέλη των Hox και polycomb πρωτεϊνών, καθώς επίσης και να δρα σαν ρυθμιστής του κυτταρικού κύκλου, λειτουργώντας ως αναστολέας του παράγοντα αδειοδότησης της αντιγραφής του DNA, CDT1. Στα πλαίσια της εργασίας αυτής, κλωνοποιήσαμε το γονίδιο της geminin στο ποντίκι το οποίο αποτελείται από 7 εξώνια και καλύπτει μία περιοχή γύρω στα 10Kb. Σχεδιάσαμε και κλωνοποιήσαμε ένα πλασμιδιακό όχημα στόχευσης για το γονίδιο της mgeminin και εισήγαμε 3 θέσεις loxP στο γενωμικό locus αυτής, με σκοπό να δημιουργήσουμε υπό συνθήκη ελλειμματικούς ποντικούς για το γονίδιό της. Χρησιμοποιώντας το συγκεκριμένο φορέα στόχευσης αδρανοποιήσαμε το γονίδιο της Geminin σε pc3 (protamine Cre 3) πολυδύναμα κύτταρα ποντικού, δημιουργώντας ετερόζυγους ES κλώνους που φέρουν το «floxed» αλληλόμορφο, καθώς επίσης και το αλληλόμορφο αγρίου τύπου. Το μεταλλαγμένο αλληλόμορφο ελέγχθηκε τόσο με PCR όσο και με ανάλυση κατά Southern για την ορθότητα του ομόλογου ανασυνδυασμού. Ταυτοποιήσαμε 15 ορθά ανασυνδυασμένους ES κλώνους, οι οποίοι μπορούν να χρησιμοποιηθούν για τη δημιουργία υπό συνθήκη knockout ποντικών για το γονίδιο της geminin, μετά από έγχυση σε B6 βλαστοκύστεις και επακόλουθη μεταφορά αυτών σε θετές μητέρες. / Geminin is a novel bifunctional molecule with a pivotal role in the processes of differentiation and cell cycle regulation, due to its neuralizing potential, its ability to interact through Hox and polycomb group members, as well as in the inhibition of cell cycle progression through protein-protein interactions with the licensing factor Cdt1. We have cloned the mouse Geminin gene, which consists of 7 exons and spans approximately a region of 10Kb. We have generated a vector and introduced 3 loxP sites in the Geminin locus, in order to create conditional knockout mice. Using this knockout construct we inactivated the geminin locus in pc3 mouse embryonic stem cells, creating heterozygous ES clones, carrying a “floxed” and a “WT” allele for geminin. The mutant allele in the targeted ES cells has been verified with Southern blotting and PCR for the correct homologous recombination events. We identified 15 correctly targeted ES clones, which can be used for the generation of conditional geminin knockout mice, upon injection into B6 blastocysts and subsequent transfer to foster mothers.

To understand the pathophysiology of hearing loss, it is necessary to identify genes responsible for embryonic development of neurosensory cells in the inner ear. The aim of this work is to clarify the role of LIM-homeodomain transcription factor ISL1 in the development of these cells. Using Cre-loxP recombination strategy, we generated a mouse line with time and site- specific deletion of Isl1 gene in NEUROD1-Cre expressing cells (Isl1 CKO). Although the early development of stato-acoustic ganglion was not affected by Isl1 deletion, at E14,5, we observed abnormalities in neuronal migration, formation of spiral ganglion and axon guidance in the Isl1 CKO cochlea. The length of the cochlear sensory epithelium was shortened by 20% as a consequence of lower proliferation activity of sensory precursor cells. Our results suggest that ISL1 is necessary for spiral ganglion formation and innervation of the Organ of Corti. Key words: transcription factor ISL1, neurons, Cre-loxP system, mouse model