RESUMO
Phosphatidyl-inositol mannosides (PIM) are glycolipids unique to mycobacteria and other related bacteria that stimulate host immune responses and are implicated in mycobacteria pathogenicity. Here, we found that the FcRγ-coupled C-type lectin receptor DCAR (dendritic cell immunoactivating receptor; gene symbol Clec4b1) is a direct receptor for PIM. Mycobacteria activated reporter cells expressing DCAR, and delipidation of mycobacteria abolished this activity. Acylated PIMs purified from mycobacteria were identified as ligands for DCAR. DCAR was predominantly expressed in small peritoneal macrophages and monocyte-derived inflammatory cells in lungs and spleen. These cells produced monocyte chemoattractant protein-1 (MCP-1) upon PIM treatment, and absence of DCAR or FcRγ abrogated MCP-1 production. Upon mycobacterial infection, Clec4b1-deficient mice showed reduced numbers of monocyte-derived inflammatory cells at the infection site, impaired IFNγ production by T cells, and an increased bacterial load. Thus, DCAR is a critical receptor for PIM that functions to promote T cell responses against mycobacteria.
Assuntos
Proteínas de Bactérias/imunologia , Lectinas Tipo C/imunologia , Fosfatidilinositóis/imunologia , Receptores Imunológicos/imunologia , Células Th1/imunologia , Animais , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium/imunologia , Infecções por Mycobacterium/imunologiaRESUMO
A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2- mutants produced in this study will be deposited in several repository organizations. KEY POINTS: ⢠All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains ⢠Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains ⢠The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Mutação , Plasmídeos , Poliploidia , Plasmídeos/genética , Edição de Genes/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces/genética , Saccharomyces cerevisiae/genética , 3-Isopropilmalato Desidrogenase/genética , 3-Isopropilmalato Desidrogenase/metabolismo , Genoma Fúngico/genéticaRESUMO
We seek to manipulate gene function here through CRISPR-Cas9 editing of cis-regulatory sequences, rather than the more typical mutation of coding regions. This approach would minimize secondary effects of cellular responses to nonsense mediated decay pathways or to mutant protein products by premature stops. This strategy also allows for reducing gene activity in cases where a complete gene knockout would result in lethality, and it can be applied to the rapid identification of key regulatory sites essential for gene expression. We tested this strategy here with genes of known function as a proof of concept, and then applied it to examine the upstream genomic region of the germline gene Nanos2 in the sea urchin, Strongylocentrotus purpuratus. We first used CRISPR-Cas9 to target established genomic cis-regulatory regions of the skeletogenic cell transcription factor, Alx1, and the TGF-ß signaling ligand, Nodal, which produce obvious developmental defects when altered in sea urchin embryos. Importantly, mutation of cis-activator sites (Alx1) and cis-repressor sites (Nodal) result in the predicted decreased and increased transcriptional output, respectively. Upon identification of efficient gRNAs by genomic mutations, we then used the same validated gRNAs to target a deadCas9-VP64 transcriptional activator to increase Nodal transcription directly. Finally, we paired these new methodologies with a more traditional, GFP reporter construct approach to further our understanding of the transcriptional regulation of Nanos2, a key gene required for germ cell identity in S. purpuratus. With a series of reporter assays, upstream Cas9-promoter targeted mutagenesis, coupled with qPCR and in situ RNA hybridization, we concluded that the promoter of Nanos2 drives strong mRNA expression in the sea urchin embryo, indicating that its primordial germ cell (PGC)-specific restriction may rely instead on post-transcriptional regulation. Overall, we present a proof-of-principle tool-kit of Cas9-mediated manipulations of promoter regions that should be applicable in most cells and embryos for which CRISPR-Cas9 is employed.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Loci Gênicos , Regiões Promotoras Genéticas/genética , Strongylocentrotus purpuratus/embriologia , Strongylocentrotus purpuratus/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Expressão Gênica , Técnicas de Inativação de Genes , Células Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/genéticaRESUMO
Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.
Assuntos
Citocromo P-450 CYP3A/genética , Edição de Genes , Glucuronosiltransferase/genética , Inativação Metabólica/genética , Animais , Técnicas de Transferência de Genes , Genoma , Humanos , Taxa de Depuração Metabólica/genética , Camundongos , Camundongos Transgênicos , RatosRESUMO
Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.
Assuntos
Ciona intestinalis/genética , Regulação da Expressão Gênica no Desenvolvimento , Genitália Masculina/crescimento & desenvolvimento , Órgãos dos Sentidos/crescimento & desenvolvimento , Animais , Ciona/genética , Ciona/crescimento & desenvolvimento , Ciona intestinalis/crescimento & desenvolvimento , Células Epiteliais/metabolismo , Genes Homeobox , Genitália Masculina/citologia , Masculino , Modelos Biológicos , Neurônios/metabolismo , Pigmentos Biológicos , Especificidade da EspécieRESUMO
BACKGROUND: Recent developments in gene editing, using transcriptional activator-like effector nucleases (TALENs), have greatly helped the generation of genetically engineered animal models. The NK3 homeobox 1 (NKX3.1) protein plays important roles in prostate development and protein production, and functions as a tumor suppressor. Recently, NKX3.1 was shown to be associated with breast cancer in humans. METHODS: Our aim was to create a new rat model to elucidate the functions of NKX3.1. To that end, we generated Nkx3.1 knockout rats using TALENs and analyzed their phenotype. TALEN-mediated Nkx3.1 knockout was confirmed by T7 endonuclease I (T7E1) assay and DNA sequencing. Prostate weight and fertility were evaluated in the knockout rats, besides determining the proportion of epithelial cells and messenger RNA (mRNA) expression of genes associated with carcinogenesis. Breast tumors were examined by histopathology. RESULTS: Results suggested Nkx3.1 knockout rats have reduced fertility, decreased prostate weights, and increased epithelial cell layers. The mRNA expression of genes related to prostate carcinogenesis, namely Ar, Akt, and Pi3k, also increased. Moreover, the Nkx3.1 knockout rats often developed malignant breast tumors. CONCLUSIONS: We, therefore, successfully created the first Nkx3.1 knockout rat model, using TALEN-mediated gene targeting, and used it to identify defects associated with Nkx3.1 deficiency, not previously observed in mice. Loss of Nkx3.1 in rats led to lower reproductive capacity, and decreased prostate weights, apart from the risk of developing breast cancer. We, thus, proposed Nkx3.1 knockout rats as reliable models for studying the role of NKX3.1 in decreased prostate weights, fertility, and breast cancer, as well as in prostate cancer.
Assuntos
Técnicas de Inativação de Genes/métodos , Proteínas de Homeodomínio/fisiologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Fertilidade , Genes Supressores de Tumor , Proteínas de Homeodomínio/genética , Masculino , Modelos Animais , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/genéticaRESUMO
Algae accumulate large amounts of lipids produced by photosynthesis, and these lipids are expected to be utilized as feedstocks for sustainable new energies, known as biodiesels. Nannochloropsis species are eukaryotic microalgae that produce high levels of lipids. However, since the production costs of algal biodiesels are higher than those of fossil fuels, the improved productivity of algal lipids by molecular breeding of algae is required for practical use. In the present study, we developed a highly efficient genome-editing system involving Platinum transcription activator-like effector nucleases (TALENs) in Nannochloropsis oceanica. Platinum TALENs codon-optimized for N. oceanica were synthesized, and their DNA-binding activity was confirmed by single-strand annealing assays in human HEK293T cells. All-in-one expression vectors for Platinum TALEN targeting the nitrate reductase gene, NoNR, and acyltransferase gene, LPAT1, were transfected into Nannochloropsis species. The introduction of each Platinum TALEN revealed high genome-editing efficiency with no detectable off-target mutations at the candidate sites in N. oceanica. By simultaneously introducing TALENs targeting two genes, we obtained double mutant strains. The loss-of-function phenotype of NoNR was also confirmed. These findings will provide an essential technology for molecular breeding in Nannochloropsis species.
Assuntos
Edição de Genes/métodos , Microalgas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/genética , Microalgas/metabolismo , Plasmídeos/genética , Estramenópilas/genética , Estramenópilas/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Transfecção/métodosRESUMO
BACKGROUND: The structure of the mouse incisor is characterized by its asymmetric accumulation of enamel matrix proteins on the labial side. The asymmetric structure originates from the patterning of the epithelial incisor placode through the interaction with dental mesenchymal cells. However, the molecular basis for the asymmetric patterning of the incisor germ is largely unknown. RESULTS: A homeobox transcription factor SIX1 was shown to be produced in the mandibular mesenchyme, and its localization patterns changed dynamically during lower incisor development. Six1-/- mice exhibited smaller lower incisor primordia than wild-type mice. Furthermore, Six1-/- mice showed enamel matrix production on both the lingual and labial sides and disturbed odontoblast maturation. In the earlier stages of development, the formation of signaling centers, the initiation knot and the enamel knot, which are essential for the morphogenesis of tooth germs, were impaired in Six1-/- embryos. Notably, Wnt signaling activity, which shows an anterior-posterior gradient, and the expression patterns of genes involved in incisor formation were altered in the mesenchyme in Six1-/- embryos. CONCLUSION: Our results indicate that Six1 is required for signaling center formation in lower incisor germs and the labial-lingual asymmetry of the lower incisors by regulating the anterior-posterior patterning of the mandibular mesenchyme.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Incisivo/embriologia , Odontoblastos/metabolismo , Odontogênese , Transdução de Sinais , Animais , Proteínas de Homeodomínio/genética , Incisivo/citologia , Camundongos , Camundongos Knockout , Odontoblastos/citologia , Germe de Dente/embriologiaRESUMO
ATF6α is an endoplasmic reticulum (ER)-embedded transcription factor which is rapidly activated by ER stress, and a major regulator of ER chaperone levels in vertebrates. We previously suggested that ATF6α occurs as a monomer, dimer and oligomer in the unstressed ER of Chinese hamster ovary cells due to the presence of two evolutionarily conserved cysteine residues in its luminal region (C467 and C618), and showed that ATF6α is reduced upon ER stress, such that only reduced monomer ATF6α is translocated to the Golgi apparatus for activation by proteolysis. However, mutagenesis analysis (C467A and C618A) revealed that the C618A mutant behaves in an unexpected manner (monomer and oligomer) during non-reducing SDS-PAGE, for reasons which remained unclear. Here, we used human colorectal carcinoma-derived HCT116 cells deficient in ATF6α and its relevant ATF6ß, and found that ATF6α dimer and oligomer are both dimers, which we designated C618-dimer and C467-dimer, respectively. We demonstrated that C467-dimer (previously considered an oligomer) behaved bigger than C618-dimer (previously considered a dimer) during non-reducing SDS-PAGE, based on their disulfide-bonded structures. Furthermore, ATF6α monomer physically associates with another ATF6α monomer in the absence of disulfide bonding, which renders two C467 residues in close proximity so that formation of C467-dimer is much easier than that of C618-dimer. In contrast, C618-dimer is more easily reduced upon ER stress. Thus, our analysis revealed that all forms of ATF6α, namely monomer, C618-dimer and C467-dimer, are activated by single reduction of a disulfide bond in response to ER stress, ensuring the rapidity of ATF6α activation.Key words: disulfide-bonded structure, endoplasmic reticulum, membrane-bound transcription factor, non-reducing SDS-PAGE, unfolded protein response.
Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Fator 6 Ativador da Transcrição/genética , Animais , Cricetinae , Cricetulus/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/metabolismo , Humanos , Chaperonas Moleculares/metabolismoRESUMO
BACKGROUND: Immune checkpoint blockade (ICB) therapy improved the prognosis of cancer patients, but general administration of ICBs occasionally induces side effects that include immune-related adverse events and tumour hyper-progression. Here, we established a protein-based system, by which endogenous expression of IC molecule in natural killer (NK) cells was transiently repressed on enhancement of their antitumour activity. METHODS: A protein-based genome modulator (GM) system is composed of a transcription activator-like effector (TALE), DNA methyltransferase and a newly identified potent cell-penetrating peptide with nuclear-trafficking property named NTP. TALE was designed to target the promoter region of the programmed cell death-1 (PD-1) gene. After culturing human NK cells in the presence of NTP-GM protein, we examined endogenous PD-1 expression and antitumour activity of the treated cells. RESULTS: NTP-GM protein efficiently downregulated PD-1 expression in NK cells with increased CpG DNA methylation in the promoter region. The antitumour activity of the treated NK cells was enhanced, and repeated intraperitoneal administrations of the treated NK cells attenuated tumour growth of programmed death-ligand 1-positive tumour cells in vivo. CONCLUSIONS: Because the incorporated NTP-GM protein was quickly degraded and negligible in the administered NK cells, the NTP-GM system could be an alternative option of an ICB without side effects.
Assuntos
Repressão Epigenética/imunologia , Proteínas de Checkpoint Imunológico/imunologia , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Animais , Humanos , Camundongos , PrognósticoRESUMO
The chordate pharynx, possessing gill slits and the endostyle, is a complex of multiple tissues that are highly organized along the anterior-posterior (AP) axis. Although Hox genes show AP coordinated expression in the pharyngeal endoderm, tissue-specific roles of these factors for establishing the regional identities within this tissue have not been demonstrated. Here, we show that Hox1 is essential for the establishment of AP axial identity of the endostyle, a major structure of the pharyngeal endoderm, in the ascidian Ciona intestinalis We found that knockout of Hox1 causes posterior-to-anterior transformation of the endostyle identity, and that Hox1 represses Otx expression and anterior identity, and vice versa. Furthermore, alteration of the regional identity of the endostyle disrupts the formation of body wall muscles, suggesting that the endodermal axial identity is essential for coordinated pharyngeal development. Our results demonstrate an essential role of Hox genes in establishment of the AP regional identity in the pharyngeal endoderm and reveal crosstalk between endoderm and mesoderm during development of chordate pharynx.
Assuntos
Endoderma/embriologia , Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular , Músculos Faríngeos/embriologia , Faringe/embriologia , Animais , Ciona intestinalis , Endoderma/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Músculos Faríngeos/efeitos dos fármacos , Faringe/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
TAp63 is an isoform of p63 gene, a p53 family gene that suppresses tumorigenesis via transcriptional regulation. TAp63 represses transcription of MYC oncogene in glioblastomas; however, its role in another MYC family gene, MYCN, has remained elusive. In this study, we showed that TAp63 repressed transcription of the MYCN gene in human cancer cells. Overexpression of TAp63 in HeLa cells suppressed MYCN expression, whereas knockdown of TAp63 had the opposite effect. By binding to exon 1 of MYCN gene, TAp63 suppressed the promoter activities of MYCN and its cis-antisense gene, NCYM. Other p53 family members, p53 and TAp73, showed lesser ability to suppress MYCN/NCYM promoter activities compared with that of TAp63. All-trans-retinoic acid (ATRA) treatment of MYCN/NCYM-amplified neuroblastoma CHP134â¯cells induced TAp63 and reduced p53 expressions, accompanied by downregulation of MYCN/NCYM expressions. Meanwhile, TAp63 knockdown inhibited ATRA-induced repression of NCYM gene expression. Blocking the p53 family binding sites by CRISPR-dCas9 system in CHP134â¯cells induced MYCN/NCYM expression and promoted apoptotic cell death. Expression levels of TAp63â¯mRNA inversely correlated with those of MYCN/NCYM expression in primary neuroblastomas, which was associated with a favorable prognosis. Collectively, TAp63 repressed MYCN/NCYM bidirectional transcription, contributing to the suppression of neuroblastoma growth.
Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Proliferação de Células/genética , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.
Assuntos
Células Clonais/metabolismo , Edição de Genes , Heterogeneidade Genética , Hipoxantina Fosforribosiltransferase/genética , Ribonucleoproteínas/genética , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Células HCT116 , Humanos , Mutagênese , RNA Guia de Cinetoplastídeos , Análise de Célula ÚnicaRESUMO
Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Animais , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodosRESUMO
Vertebrates have unique head structures that are mainly composed of the central nervous system, the neural crest, and placode cells. These head structures are brought about initially by the neural induction between the organizer and the prospective neuroectoderm at early gastrula stage. Purinergic receptors are activated by nucleotides released from cells and influence intracellular signaling pathways, such as phospholipase C and adenylate cyclase signaling pathways. As P2Y receptor is vertebrate-specific and involved in head formation, we expect that its emergence may be related to the acquisition of vertebrate head during evolution. Here, we focused on the role of p2ry4 in early development in Xenopus laevis and found that p2ry4 was required for the establishment of the head organizer during neural induction and contributed to head formation. We showed that p2ry4 was expressed in the head organizer region and the prospective neuroectoderm at early gastrula stage, and was enriched in the head components. Disruption of p2ry4 function resulted in the small head phenotype and the reduced expression of marker genes specific for neuroectoderm and neural border at an early neurula stage. Furthermore, we examined the effect of p2ry4 disruption on the establishment of the head organizer and found that a reduction in the expression of head organizer genes, such as dkk1 and cerberus, and p2ry4 could also induce the ectopic expression of these marker genes. These results suggested that p2ry4 plays a key role in head organizer formation. Our study demonstrated a novel role of p2ry4 in early head development.
Assuntos
Cabeça/crescimento & desenvolvimento , Receptores Purinérgicos P2/metabolismo , Xenopus laevis/embriologia , AnimaisRESUMO
Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells.
Assuntos
Alelos , Sistemas CRISPR-Cas/genética , Edição de Genes , Genoma Humano , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Homozigoto , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Rad51 Recombinase/genética , Transcrição Gênica/efeitos dos fármacos , Ácido Valproico/farmacologiaRESUMO
Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.
Assuntos
Proteínas de Homeodomínio/genética , Morfogênese/genética , Escoliose/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Adolescente , Animais , Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/biossíntese , Humanos , Polimorfismo de Nucleotídeo Único , Escoliose/patologia , Fatores de Transcrição/biossíntese , Proteínas Wnt/biossíntese , Via de Sinalização Wnt/genética , Proteína Wnt-5a , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.
Assuntos
Ciona intestinalis/citologia , Ciona intestinalis/genética , Células Germinativas/citologia , Mutagênese/genética , Regeneração , Animais , Animais Geneticamente Modificados , Sequência de Bases , Eletroporação , Técnicas de Inativação de Genes , Genoma , Células Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Masculino , Metamorfose Biológica , Mutação/genética , Taxa de Mutação , Especificidade de Órgãos , Óvulo/citologia , Espermatozoides/citologia , Cauda , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Genome editing includes various edits of the genome, such as short insertions and deletions, substitutions, and chromosomal rearrangements including inversions, duplications, and translocations. These variations are based on single or multiple DNA double-strand break (DSB)-triggered in cellulo repair machineries. In addition to these "conventional" genome editing strategies, tools enabling customized, site-specific recognition of particular nucleic acid sequences have been coming into wider use; for example, single base editing without DSB introduction, epigenome editing with recruitment of epigenetic modifiers, transcriptome engineering using RNA editing systems, and in vitro detection of specific DNA and RNA sequences. In this review, we provide a quick overview of the current state of genome editing and related technologies that multilaterally contribute to cancer science.
Assuntos
Edição de Genes/métodos , Neoplasias/genética , Pesquisa Biomédica , Epigenômica , Perfilação da Expressão Gênica , Humanos , Análise de Sequência de RNARESUMO
Interleukin (IL)-11 belongs to the members of the IL-6 family of cytokines and is involved in a variety of biological responses, including hematopoiesis, bone development, and carcinogenesis. However, the cellular sources of IL-11 and regulation of IL-11 expression under physiological and pathological conditions are not fully understood. One of the causes to prevent characterization of IL-11 in vivo is due to the lack of reliable antibodies that detect IL-11 by immunohistochemistry. Moreover, although mice lacking Il11ra have been generated and extensively characterized, Il11-deficient mice have not been characterized yet. Here we generated two anti-IL-11 antibodies that blocked biological activities of IL-11 and detected IL-11 by immunohistochemistry, respectively. One clone of anti-IL-11 antibodies blocked IL-11-, but not IL-6-induced cell proliferation and IL-11-induced phosphorylation of STAT3 of an IL-11-dependent cell line. Moreover, we used recently established Il11-deficient mice to test the specificity of anti-IL-11 antibodies for immunohistochemistry. Another clone of anti-IL-11 antibodies stained stromal cells surrounding tumors of the colon of wild-type, but not Il11-deficient mice following treatment with Azoxymethane plus dextran sulfate sodium. Together, these newly developed anti-IL-11 antibodies provide a better understanding of the functions of IL-11 in vivo under various physiological and pathological conditions.