Generation and characterization of Ccdc28b mutant mice links the Bardet-Biedl associated gene with mild social behavioral phenotypes.

CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.

Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.

Bardet-Biedl syndrome: Is it only cilia dysfunction?

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic disorder, characterized by both congenital and late onset defects. From the analysis of the mutational burden in patients to the functional characterization of the BBS proteins, this syndrome has become a model for both understanding oligogenic patterns of inheritance and the biology of a particular cellular organelle: the primary cilium. Here we briefly review the genetics of BBS to then focus on the function of the BBS proteins, not only in the context of the cilium but also highlighting potential extra-ciliary roles that could be relevant to the etiology of the disorder. Finally, we provide an overview of how the study of this rare syndrome has contributed to the understanding of cilia biology and how this knowledge has informed on the cellular basis of different clinical manifestations that characterize BBS and the ciliopathies.

Optimization of transgenesis conditions for the generation of CXCL2-luciferase reporter mice line

Background: Transgenesis by microinjection has been widely used for the generation of different mouse models. Different variables of the procedure may critically affect the efficiency of the process. A DNA construction that carries the CXCL2 promoter gene and firefly luciferase has been used to optimize aspects of the procedure. Three different concentrations (0.5, 1.0 and 4.0 ng/µl) of the DNA construction to microinject a total of 1981 zygotes has been tested. Intact/injected embryos, pregnancy and birth rate, survival of pups 7 days after birth, number of transgenic pups and overall transgenic efficiency was registered and analyzed by Z test of proportions for each group. Results: A total of seven transgenic founders were detected for the three DNA concentrations used, 1 in 46 alive pups in the 0.5 ng/µl group, 5 in 38 alive pups in the 1 ng/µl group and 1 in 21 alive pups in the 4 ng/µl group ( p < 0.1). The overall transgenic efficiency was higher for the 1 ng/µl concentration, with a transgenic rate of 13.2%. Conclusions: In conclusion, we have selected the best operative conditions to maximize the transgenesis efficiency. Furthermore, the transgenic lines developed could be used as a reporter model of innate immunity activation with many different applications in the fields of immunology, cancer and neurodegenerative diseases.

Characterization of CCDC28B reveals its role in ciliogenesis and provides insight to understand its modifier effect on Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that is generally inherited in an autosomal recessive fashion. However, in some families, trans mutant alleles interact with the primary causal locus to modulate the penetrance and/or the expressivity of the phenotype. CCDC28B (MGC1203) was identified as a second site modifier of BBS encoding a protein of unknown function. Here we report the first functional characterization of this protein and show it affects ciliogenesis both in cultured cells and in vivo in zebrafish. Consistent with this biological role, our in silico analysis shows that the presence of CCDC28B homologous sequences is restricted to ciliated metazoa. Depletion of Ccdc28b in zebrafish results in defective ciliogenesis and consequently causes a number of phenotypes that are characteristic of BBS and other ciliopathy mutants including hydrocephalus, left-right axis determination defects and renal function impairment. Thus, this work reports CCDC28B as a novel protein involved in the process of ciliogenesis whilst providing functional insight into the cellular basis of its modifier effect in BBS patients.

Eficiencia de la transgenesis murina en el institut pasteur de Montevideo / /Murine transgenesis techniques efficiency of institute pasteur Montevideo

Los animales transgénicos son empleados en investigación biomédica para estudiar la función y regulación de los genes, como modelos de enfermedades humanas y para ensayar estrategias terapéuticas. Asimismo, la transgénesis es aplicada en animales productivos para la generación de proteínas recombinantes y la mejora animal, y para la realización de xenotrasplantes. La Unidad de Animales Transgénicos y de Experimentación del Institut Pasteur de Montevideo produce ratones transgénicos a demanda para investigadores locales y de la región. El objetivo de este trabajo es mostrar la eficiencia durante la puesta a punto de alguna de las técnicas empleadas: (i) microinyección pronuclear de ADN (Tg clásica); (ii) transgénesis mediada por lentivirus (LV); y (iii) microinyección de células madre embrionarias (ESC) en blastocistos. Los parámetros evaluados fueron: sobrevida embrionaria 30 min post-inyección, tasa de preñez, de nacimiento, de transgénesis y tasa global de transgénesis. La sobrevida embrionaria resultó en 54,6%, 78,8% y 87,8% para Tg clásica, ESC y LV, respectivamente. El número de embriones GFP (proteína verde fluorescente) + inyectados con LV resultó 42,6%. La tasa de preñez fue 54,8% y 25,0%; la tasa de nacimiento fue 21,3% y 24,0%; la tasa de transgénesis fue 4,5% y 24,1%; y la tasa de transgénesis global fue 0,2% y 0,8%, para Tg clásica y ESC, respectivamente. En suma, se produjeron 21 animales fundadores por Tg clásica, 7 animales quiméricos por inyección de ESC, y más de un 50% de embriones resultaron GFP+ con la técnica de inyección de LV. Las técnicas empleadas resultaron efectivas para obtener los modelos transgénicos solicitados. CEUA Nº 01-012.

Transgenic animals are used in biomedical research to study gene function and regulation, as human disease models, and to test therapeutic strategies. Moreover, transgenic farm animals are used for recombinant protein production, sanitary improvement, and xenotransplantation. The Transgenic and Experimental Animal Unit of Institut Pasteur Montevideo produces transgenic mice for local and regional researchers. The objective of this work is to show the efficiency of transgenesis techniques during their set up at the unit: (i) DNA pronuclear microinjection (classical Tg); (ii) lentiviral-mediated transgenesis (LV); and (iii) embryonic stem cell (ESC) microinjection in blastocysts. The parameters evaluated were: embryo survival 30 min post-injection, pregnancy rate, birth rate, transgenesis rate, and global transgenesis rate. Embryo survival was 54.6%, 78.8% and 87.8% for classical Tg, ESC and LV, respectively. The number of GFP+ embryos injected with LV was 42.6%. Pregnancy rate was 54.8% and 25.0%; birth rate was 21.3% and 24.0%; transgenesis rate was 4.5% and 24.1%; and global transgenesis rate was 0.2% and 0.8%, for classical Tg and ESC, respectively. In total, 21 founder animals were produced by classical Tg, 7 chimeric animals by ESC injection, and more than 50% GFP+ embryos were obtained using LV injection. The techniques used at the unit were effective to obtain the required transgenic models.

Eficiencia de la transgenesis murina en el institut pasteur de Montevideo / /Murine transgenesis techniques efficiency of institute pasteur Montevideo

Los animales transgénicos son empleados en investigación biomédica para estudiar la función y regulación de los genes, como modelos de enfermedades humanas y para ensayar estrategias terapéuticas. Asimismo, la transgénesis es aplicada en animales productivos para la generación de proteínas recombinantes y la mejora animal, y para la realización de xenotrasplantes. La Unidad de Animales Transgénicos y de Experimentación del Institut Pasteur de Montevideo produce ratones transgénicos a demanda para investigadores locales y de la región. El objetivo de este trabajo es mostrar la eficiencia durante la puesta a punto de alguna de las técnicas empleadas: (i) microinyección pronuclear de ADN (Tg clásica); (ii) transgénesis mediada por lentivirus (LV); y (iii) microinyección de células madre embrionarias (ESC) en blastocistos. Los parámetros evaluados fueron: sobrevida embrionaria 30 min post-inyección, tasa de preñez, de nacimiento, de transgénesis y tasa global de transgénesis. La sobrevida embrionaria resultó en 54,6%, 78,8% y 87,8% para Tg clásica, ESC y LV, respectivamente. El número de embriones GFP (proteína verde fluorescente) + inyectados con LV resultó 42,6%. La tasa de preñez fue 54,8% y 25,0%; la tasa de nacimiento fue 21,3% y 24,0%; la tasa de transgénesis fue 4,5% y 24,1%; y la tasa de transgénesis global fue 0,2% y 0,8%, para Tg clásica y ESC, respectivamente. En suma, se produjeron 21 animales fundadores por Tg clásica, 7 animales quiméricos por inyección de ESC, y más de un 50% de embriones resultaron GFP+ con la técnica de inyección de LV. Las técnicas empleadas resultaron efectivas para obtener los modelos transgénicos solicitados. CEUA Nº 01-012.(AU)

Transgenic animals are used in biomedical research to study gene function and regulation, as human disease models, and to test therapeutic strategies. Moreover, transgenic farm animals are used for recombinant protein production, sanitary improvement, and xenotransplantation. The Transgenic and Experimental Animal Unit of Institut Pasteur Montevideo produces transgenic mice for local and regional researchers. The objective of this work is to show the efficiency of transgenesis techniques during their set up at the unit: (i) DNA pronuclear microinjection (classical Tg); (ii) lentiviral-mediated transgenesis (LV); and (iii) embryonic stem cell (ESC) microinjection in blastocysts. The parameters evaluated were: embryo survival 30 min post-injection, pregnancy rate, birth rate, transgenesis rate, and global transgenesis rate. Embryo survival was 54.6%, 78.8% and 87.8% for classical Tg, ESC and LV, respectively. The number of GFP+ embryos injected with LV was 42.6%. Pregnancy rate was 54.8% and 25.0%; birth rate was 21.3% and 24.0%; transgenesis rate was 4.5% and 24.1%; and global transgenesis rate was 0.2% and 0.8%, for classical Tg and ESC, respectively. In total, 21 founder animals were produced by classical Tg, 7 chimeric animals by ESC injection, and more than 50% GFP+ embryos were obtained using LV injection. The techniques used at the unit were effective to obtain the required transgenic models.(AU)

Direct role of Bardet-Biedl syndrome proteins in transcriptional regulation.

Primary cilia are conserved organelles that play crucial roles as mechano- and chemosensors, as well as transducing signaling cascades. Consequently, ciliary dysfunction results in a broad range of phenotypes: the ciliopathies. Bardet-Biedl syndrome (BBS), a model ciliopathy, is caused by mutations in 16 known genes. However, the biochemical functions of the BBS proteins are not fully understood. Here we show that the BBS7 protein (localized in the centrosomes, basal bodies and cilia) probably has a nuclear role by virtue of the presence of a biologically confirmed nuclear export signal. Consistent with this observation, we show that BBS7 interacts physically with the polycomb group (PcG) member RNF2 and regulate its protein levels, probably through a proteasome-mediated mechanism. In addition, our data supports a similar role for other BBS proteins. Importantly, the interaction with this PcG member is biologically relevant because loss of BBS proteins leads to the aberrant expression of endogenous RNF2 targets in vivo, including several genes that are crucial for development and for cellular and tissue homeostasis. Our data indicate a hitherto unappreciated, direct role for the BBS proteins in transcriptional regulation and potentially expand the mechanistic spectrum that underpins the development of ciliary phenotypes in patients.

Ciliary biology: understanding the cellular and genetic basis of human ciliopathies.

Motile cilia have long been known to play a role in processes such as cell locomotion and fluid movement whereas the functions of primary cilia have remained obscure until recent years. To date, ciliary dysfunction has been shown to be causally linked to a number of clinical manifestations that characterize the group of human disorders known as ciliopathies. This classification reflects a common or shared cellular basis and implies that it is possible to associate a series of different human conditions with ciliary dysfunction, which allows gaining insight into the cellular defect in disorders of unknown etiology solely based on phenotypic observations. Furthermore, to date we know that the cilium participates in a number of biological processes ranging from chemo- and mechanosensation to the transduction of a growing list of paracrine signaling cascades that are critical for the development and maintenance of different tissues and organs. Consequently, the primary cilium has been identified as a key structure necessary to regulate and maintain cellular and tissue homeostasis and thus its study is providing significant information to understand the pathogenesis of the different phenotypes that characterize these human conditions. Finally, the similarities between different ciliopathies at the phenotypic level are proving to be due to their shared cellular defect and also their common genetic basis. To this end, recent studies are showing that mutations in a given ciliary gene often appear involved in the pathogenesis of more than one clinical entity, complicating their genetic dissection, and hindering our ability to generate accurate genotype-phenotype correlations.