RESUMO
Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, although successful lung formation using interspecies chimeric animals has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. These findings provide useful insights to overcome the barrier of species-specific developmental timing to generate functional lungs in interspecies chimeras.
Assuntos
Células-Tronco Pluripotentes , Ratos , Camundongos , Animais , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Blastocisto , Pulmão , Células Epiteliais , Modelos Animais de DoençasRESUMO
Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.
Assuntos
Proteínas Nucleares , Complexo Sinaptonêmico , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Espermatócitos/metabolismo , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
C3-positive reactive astrocytes play a neurotoxic role in various neurodegenerative diseases. However, the mechanisms controlling C3-positive reactive astrocyte induction are largely unknown. We found that the length of the primary cilium, a cellular organelle that receives extracellular signals was increased in C3-positive reactive astrocytes, and the loss or shortening of primary cilium decreased the count of C3-positive reactive astrocytes. Pharmacological experiments suggested that Ca2+ signalling may synergistically promote C3 expression in reactive astrocytes. Conditional knockout (cKO) mice that specifically inhibit primary cilium formation in astrocytes upon drug stimulation exhibited a reduction in the proportions of C3-positive reactive astrocytes and apoptotic cells in the brain even after the injection of lipopolysaccharide (LPS). Additionally, the novel object recognition (NOR) score observed in the cKO mice was higher than that observed in the neuroinflammation model mice. These results suggest that the primary cilium in astrocytes positively regulates C3 expression. We propose that regulating astrocyte-specific primary cilium signalling may be a novel strategy for the suppression of neuroinflammation.
Assuntos
Astrócitos , Cílios , Camundongos Knockout , Animais , Astrócitos/metabolismo , Astrócitos/efeitos dos fármacos , Cílios/metabolismo , Cílios/efeitos dos fármacos , Camundongos , Complemento C3/metabolismo , Camundongos Endogâmicos C57BL , Lipopolissacarídeos/farmacologia , Apoptose/efeitos dos fármacosRESUMO
Genetic lineage-tracing techniques are powerful tools for studying specific cell populations in development and pathogenesis. Previous techniques have mainly involved systems for tracing a single gene, which are limited in their ability to facilitate direct comparisons of the contributions of different cell lineages. We have developed a new combinatorial system for tracing all three germ layers using self-cleaving 2A peptides and multiple site-specific recombinases (SSRs). In the resulting TRiCK (TRiple Coloured germ layer Knock-in) mice, the three germ layers are conditionally and simultaneously labelled with distinct fluorescent proteins via embryogenesis. We show that previously reported ectopic expressions of lineage markers are the outcome of secondary gene expression. The results presented here also indicate that the commitment of caudal axial stem cells to neural or mesodermal fate proceeds without lineage fluctuations, contrary to the notion of their bi-potency. Moreover, we developed IMES, an optimized tissue clearing method that is highly compatible with a variety of fluorescent proteins and immunostaining, and the combined use of TRiCK mice and IMES can facilitate comprehensive analyses of dynamic contributions of all three germ layers.
Assuntos
Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Camadas Germinativas/citologia , Animais , Encéfalo/metabolismo , Cruzamentos Genéticos , DNA Nucleotidiltransferases/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endotélio Vascular/citologia , Feminino , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Coração/embriologia , Humanos , Imageamento Tridimensional , Fígado/embriologia , Masculino , Mesoderma/citologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/citologia , Placa Neural/citologiaRESUMO
TRA98 is a rat monoclonal antibody (mAb) which recognizes a specific antigen in the nuclei of germ cells. mAb TRA98 has been used to understand the mechanism of germ cell development and differentiation in many studies. In mice, the antigen recognized by mAb TRA98 or GCNA1 has been reported to be a GCNA gene product, but despite the demonstration of the immunoreactivity of this mAb in human testis and sperm in 1997, the antigen in humans remains unknown, as of date. To identify the human antigen recognized by mAb TRA98, a human comprehensive wet protein array was developed containing 19,446 proteins derived from human cDNAs. Using this array, it was found that the antigen of mAb TRA98 is not a GCNA gene product, but nuclear factor-κB activating protein (NKAP). In mice, mAb TRA98 recognized both the GCNA gene product and NKAP. Furthermore, conditional knockout of Nkap in mice revealed a phenotype of Sertoli cell-only syndrome. Although NKAP is a ubiquitously expressed protein, NKAP recognized by mAb TRA98 in mouse testis was SUMOylated. These results suggest that NKAP undergoes modifications, such as SUMOylation in the testis, and plays an important role in spermatogenesis.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos/metabolismo , Células Germinativas/metabolismo , Análise Serial de Proteínas , Animais , Humanos , Masculino , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Testículo/metabolismoRESUMO
Random gene trapping is the application of insertional mutagenesis techniques that are conventionally used to inactivate protein-coding genes in mouse embryonic stem (ES) cells. Transcriptionally silent genes are not effectively targeted by conventional random gene trapping techniques, thus we herein developed an unbiased poly (A) trap (UPATrap) method using a Tol2 transposon, which preferentially integrated into active genes rather than silent genes in ES cells. To achieve efficient trapping at transcriptionally silent genes using random insertional mutagenesis in ES cells, we generated a new diphtheria toxin (DT)-mediated trapping vector, DTrap that removed cells, through the expression of DT that was induced by the promoter activity of the trapped genes, and selected trapped clones using the neomycin-resistance gene of the vector. We found that a double-DT, the dDT vector, dominantly induced the disruption of silent genes, but not active genes, and showed more stable integration in ES cells than the UPATrap vector. The dDT vector disrupted differentiated cell lineage genes, which were silent in ES cells, and labeled trapped clone cells by the expression of EGFP upon differentiation. Thus, the dDT vector provides a systematic approach to disrupt silent genes and examine the cellular functions of trapped genes in the differentiation of target cells and development.
Assuntos
Elementos de DNA Transponíveis , Toxina Diftérica/genética , Marcação de Genes/métodos , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Mutagênese , Mutagênese InsercionalRESUMO
In mammals, more than 2000 genes are specifically or abundantly expressed in testis, but gene knockout studies revealed several are not individually essential for male fertility. Tesmin (Metallothionein-like 5; Mtl5) was originally reported as a testis-specific transcript that encodes a member of the cysteine-rich motif containing metallothionein family. Later studies showed that Tesmin has two splicing variants and both are specifically expressed in male and female germ cells. Herein, we clarified that the long (Tesmin-L) and short (Tesmin-S) transcript forms start expressing from spermatogonia and the spermatocyte stage, respectively, in testis. Furthermore, while Tesmin-deficient female mice are fertile, male mice are infertile due to arrested spermatogenesis at the pachytene stage. We were able to rescue the infertility with a Tesmin-L transgene, where we concluded that TESMIN-L is critical for meiotic completion in spermatogenesis and indispensable for male fertility.
Assuntos
Fertilidade/genética , Metalotioneína/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Animais , Azoospermia/congênito , Azoospermia/genética , Azoospermia/metabolismo , Células COS , Chlorocebus aethiops , Masculino , Meiose/genética , Metalotioneína/genética , Camundongos , Camundongos Knockout , Espermatócitos/metabolismo , Espermatogônias/metabolismoRESUMO
Mitochondrial dysfunction in the nigrostriatal dopaminergic system is a critical hallmark of Parkinson's disease (PD). Mitochondrial toxins produce cellular and behavioural dysfunctions resembling those in patients with PD Causative gene products for familial PD play important roles in mitochondrial function. Therefore, targeting proteins that regulate mitochondrial integrity could provide convincing strategies for PD therapeutics. We have recently identified a novel 13-kDa protein (p13) that may be involved in mitochondrial oxidative phosphorylation. In the current study, we examine the mitochondrial function of p13 and its involvement in PD pathogenesis using mitochondrial toxin-induced PD models. We show that p13 overexpression induces mitochondrial dysfunction and apoptosis. p13 knockdown attenuates toxin-induced mitochondrial dysfunction and apoptosis in dopaminergic SH-SY5Y cells via the regulation of complex I. Importantly, we generate p13-deficient mice using the CRISPR/Cas9 system and observe that heterozygous p13 knockout prevents toxin-induced motor deficits and the loss of dopaminergic neurons in the substantia nigra. Taken together, our results suggest that manipulating p13 expression may be a promising avenue for therapeutic intervention in PD.
Assuntos
Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Doença de Parkinson/genética , Transtornos Parkinsonianos/genética , Animais , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Fosforilação Oxidativa , Estresse Oxidativo/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologiaRESUMO
More than 1000 genes are predicted to be predominantly expressed in mouse testis, yet many of them remain unstudied in terms of their roles in spermatogenesis and sperm function and their essentiality in male reproduction. Since individually indispensable factors can provide important implications for the diagnosis of genetically related idiopathic male infertility and may serve as candidate targets for the development of nonhormonal male contraceptives, our laboratories continuously analyze the functions of testis-enriched genes in vivo by generating knockout mouse lines using the CRISPR/Cas9 system. The dispensability of genes in male reproduction is easily determined by examining the fecundity of knockout males. During our large-scale screening of essential factors, we knocked out 30 genes that have a strong bias of expression in the testis and are mostly conserved in mammalian species including human. Fertility tests reveal that the mutant males exhibited normal fecundity, suggesting these genes are individually dispensable for male reproduction. Since such functionally redundant genes are of diminished biological and clinical significance, we believe that it is crucial to disseminate this list of genes, along with their phenotypic information, to the scientific community to avoid unnecessary expenditure of time and research funds and duplication of efforts by other laboratories.
Assuntos
Sistemas CRISPR-Cas , Fertilidade/genética , Edição de Genes , Regulação da Expressão Gênica/fisiologia , Testículo/metabolismo , Animais , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , TranscriptomaRESUMO
About 10% of male infertile patients show abnormalities in spermatogenesis. The microdeletion of azoospermia factor a (AZFa) region of the Y chromosome is thought to be a cause of spermatogenic failure. However, candidate gene responsible for the spermatogenic failure in AZFa deleted patients has not been elucidated yet. Using mice, we explored the function of Ddx3y, a strong candidate gene in the Azfa region, and Ddx3x, a Ddx3y paralog on the X chromosome, in spermatogenesis. We first generated Ddx3y KO male mice using CRISPR/Cas9 and found that the Ddx3y KO male mice show normal spermatogenesis, produce morphologically normal spermatozoa, and sire healthy offspring. Because Ddx3x KO males were embryonic lethal, we next generated chimeric mice, which contain Ddx3x and Ddx3y double KO (dKO) germ cells, and found that the dKO germ cells can differentiate into spermatozoa and transmit their mutant alleles to offspring by normal mating. We conclude that Ddx3x and Ddx3y are dispensable for spermatogenesis at least in mice. Unlike human, mice have an additional Ddx3y paralog D1pas1, that has been reported to be essential for spermatogenesis. These findings suggest that human and mouse DDX3 related proteins have distinct differences in their functions.
Assuntos
Azoospermia/genética , RNA Helicases DEAD-box/genética , Fertilidade/genética , Células Germinativas/metabolismo , Antígenos de Histocompatibilidade Menor/genética , RNA Helicases/genética , Animais , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , RNA Helicases/metabolismo , Homologia de Sequência , Espermatogênese/genética , Espermatozoides/metabolismoRESUMO
Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.
Assuntos
Fertilidade/genética , Testículo/metabolismo , Animais , Evolução Biológica , Sistemas CRISPR-Cas , Feminino , Fertilização , Engenharia Genética , Genômica , Masculino , Camundongos , Camundongos Knockout , EspermatogêneseRESUMO
Acrosin, the trypsin-like serine protease in the sperm acrosome, was long viewed as a key enzyme required for zona pellucida penetration to fertilize eggs. However, gene disruption experiments in mice surprisingly showed that acrosin-disrupted males were fertile. Thus, the acrosin was considered to be not an essential enzyme for fertilization in mice. However, the involvement of acrosin in fertilization has been suggested in various species such as rat, bull, and pig. Moreover, it has been reported that serine protease (including acrosin) activity in mice is significantly weaker compared to other species, including rats. We analyzed the role of acrosin by disrupting the rat acrosin gene. It was found that, unlike in mice, acrosin was almost the sole source of serine protease in rat spermatozoa. Nevertheless, the acrosin-disrupted males were not infertile. However, the litter size from acrosin-disrupted males was decreased compared to heterozygous mutant rats. Further investigation using an in vitro fertilization system revealed that the acrosin-disrupted spermatozoa possessed an equal ability to penetrate the zona pellucida with wild-type spermatozoa, but the cumulus cell dispersal was slower compared to wild-type and heterozygous spermatozoa. This delay was presumed to be the cause of the small litter size of acrosin-disrupted male rats.
Assuntos
Acrosina/metabolismo , Células do Cúmulo/fisiologia , Fertilização/fisiologia , Espermatozoides/fisiologia , Acrosina/genética , Animais , Feminino , Fertilização in vitro , Deleção de Genes , Regulação da Expressão Gênica , Masculino , RatosRESUMO
Using transgenic mice with spermatozoa expressing enhanced green fluorescent protein in their acrosome and red fluorescent protein in their midpiece mitochondria, we followed the behavior of spermatozoa within the female genital tract after natural mating. When examined 15 min after coitus, many spermatozoa were around the opening of the uterotubal junction. Spermatozoa that entered the uterotubal junction were seemingly not moving, yet they steadily migrated toward the isthmus at a speed only time-lapse video recording could demonstrate. Many spermatozoa reaching the lower isthmus were motile. The site where spermatozoa attached and detached from the isthmus epithelium shifted from the lower to the upper segment of the isthmus with time. Virtually all the live spermatozoa within the lower isthmus were acrosome intact, whereas many of the actively motile spermatozoa in the upper isthmus were acrosome reacted. As far as we could observe, all the spermatozoa we found within the lumen of the ampulla and the cumulus oophorus were acrosome reacted. Even though we saw only a very few spermatozoa within the ampulla during fertilization, all were associated with, or were already within, oocytes, indicating that mouse fertilization in vivo is extremely efficient.
Assuntos
Fertilização , Espermatozoides/fisiologia , Reação Acrossômica , Animais , Feminino , Masculino , Camundongos , Camundongos Transgênicos , OviductosRESUMO
BACKGROUND: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. RESULTS: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. CONCLUSIONS: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.
Assuntos
Marcação de Genes , Técnicas de Transferência de Genes , Animais , Células-Tronco Embrionárias , Feminino , Injeções/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
SPACA1 is a membrane protein that localizes in the equatorial segment of spermatozoa in mammals and is reported to function in sperm-egg fusion. We produced a Spaca1 gene-disrupted mouse line and found that the male mice were infertile. The cause of this sterility was abnormal shaping of the sperm head reminiscent of globozoospermia in humans. Disruption of Spaca1 led to the disappearance of the nuclear plate, a dense lining of the nuclear envelope facing the inner acrosomal membrane. This coincided with the failure of acrosomal expansion during spermiogenesis and resulted in the degeneration and disappearance of the acrosome in mature spermatozoa. Thus, these findings clarify part of the cascade leading to globozoospermia.
Assuntos
Infertilidade Masculina/genética , Isoantígenos/genética , Proteínas de Plasma Seminal/genética , Cabeça do Espermatozoide/patologia , Espermatozoides/anormalidades , Acrossomo/metabolismo , Acrossomo/fisiologia , Animais , Forma Celular/genética , Expressão Gênica , Infertilidade Masculina/patologia , Isoantígenos/metabolismo , Isoantígenos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Proteínas de Plasma Seminal/metabolismo , Proteínas de Plasma Seminal/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologia , Espermatozoides/ultraestrutura , Distribuição TecidualRESUMO
Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification.
Assuntos
Células-Tronco Embrionárias/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Germinativas/citologia , Células Germinativas/enzimologia , Sistema de Sinalização das MAP Quinases , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Espermatogênese/efeitos dos fármacos , Transplante de Células-Tronco , Tretinoína/farmacologiaRESUMO
Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.
Assuntos
Sistemas CRISPR-Cas/genética , Espermatogênese/fisiologia , Animais , Masculino , Camundongos , Motilidade dos Espermatozoides/genética , Motilidade dos Espermatozoides/fisiologia , Espermatogênese/genéticaRESUMO
Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.
Assuntos
Bancos de Espécimes Biológicos , Células-Tronco Embrionárias/metabolismo , Homozigoto , Mutação/genética , Bancos de Tecidos , Animais , Células-Tronco Embrionárias/citologia , Genômica , Gentamicinas/farmacologia , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas/genética , Puromicina/farmacologia , Proteínas de Ligação a RNARESUMO
Gene-knockout animal models with organ-deficient phenotypes used for blastocyst complementation are generally not viable. Animals need to be maintained as heterozygous mutants, and homozygous mutant embryos yield only one-fourth of all embryos. In this study, we generated organ-deficient embryos using the CRISPR-Cas9-sgRNAms system that induces cell death with a single-guide RNA (sgRNAms) targeting multiple sites in the genome. The Cas9-sgRNAms system interrupted cell proliferation and induced cell ablation in vitro. The mouse model had Cas9 driven by the Foxn1 promoter with a ubiquitous expression cassette of sgRNAms at the Rosa26 locus (Foxn1Cas9; Rosa26_ms). It showed an athymic phenotype similar to that of nude mice but was not hairless. Eventually, a rat cell-derived thymus in an interspecies chimera was generated by blastocyst complementation of Foxn1Cas9; Rosa26_ms mouse embryos with rat embryonic stem cells. Theoretically, a half of the total embryos has the Cas9-sgRNAms system because Rosa26_ms could be maintained as homozygous.
Assuntos
Sistemas CRISPR-Cas , Fatores de Transcrição Forkhead , RNA Guia de Sistemas CRISPR-Cas , Animais , Camundongos , Ratos , RNA Guia de Sistemas CRISPR-Cas/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Timo/metabolismo , Modelos Animais , Blastocisto/metabolismoRESUMO
Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.