RESUMO
The RIKEN BioResource Research Center (BRC) was established in 2001 as a comprehensive biological resource center in Japan. The Experimental Animal Division, one of the BRC infrastructure divisions, has been designated as the core facility for mouse resources within the National BioResource Project (NBRP) by the Japanese government since FY2002. Our activities regarding the collection, preservation, quality control, and distribution of mouse resources have been supported by the research community, including evaluations and guidance on advancing social and research needs, as well as the operations and future direction of the BRC. Expenditure for collection, preservation, and quality-control operations of the BRC, as a national core facility, has been funded by the government, while distribution has been separately funded by users' reimbursement fees. We have collected over 9000 strains created mainly by Japanese scientists including Nobel laureates and researchers in cutting-edge fields and distributed mice to 7000 scientists with 1500 organizations in Japan and globally. Our users have published 1000 outstanding papers and a few dozen patents. The collected mouse resources are accessible via the RIKEN BRC website, with a revised version of the searchable online catalog. In addition, to enhance the visibility of useful strains, we have launched web corners designated as the "Mouse of the Month" and "Today's Tool and Model." Only high-demand strains are maintained in live colonies, while other strains are cryopreserved as embryos or sperm to achieve cost-effective management. Since 2007, the RIKEN BRC has built up a back-up facility in the RIKEN Harima branch to protect the deposited strains from disasters. Our mice have been distributed with high quality through the application of strict microbial and genetic quality control programs that cover a globally accepted pathogens list and mutated alleles generated by various methods. Added value features, such as information about users' publications, standardized phenotyping data, and genome sequences of the collected strains, are important to facilitate the use of our resources. We have added and disseminated such information in collaboration with the NBRP Information Center and the NBRP Genome Information Upgrading Program. The RIKEN BRC has participated in international mouse resource networks such as the International Mouse Strain Resource, International Mouse Phenotyping Consortium, and Asian Mouse Mutagenesis and Resource Association to facilitate the worldwide use of high-quality mouse resources, and as a consequence it contributes to reproducible life science studies and innovation around the globe.
Assuntos
Programas Governamentais , Centros de Informação , Camundongos , Animais , Genoma , Japão , Camundongos/genéticaRESUMO
Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , ZigotoRESUMO
Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 em1(CreERT2)Utr , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 em1(CreERT2)Utr ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 em1(CreERT2)Utr is beneficial for studying female and male germ cell development.
Assuntos
Linhagem da Célula , RNA Helicases DEAD-box/genética , Técnicas de Introdução de Genes/métodos , Células Germinativas/metabolismo , Integrases/genética , Animais , RNA Helicases DEAD-box/metabolismo , Feminino , Células Germinativas/citologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras GenéticasRESUMO
Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2-dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers.
Assuntos
Técnicas de Reprogramação Celular/métodos , Proteínas de Ligação a DNA , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Complexo Repressor Polycomb 2 , Receptores de Estrogênio , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Fator 4 Semelhante a Kruppel , Camundongos , Complexo Repressor Polycomb 2/biossíntese , Complexo Repressor Polycomb 2/genética , Proteínas de Ligação a RNA , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 â¼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.
Assuntos
Agenesia do Corpo Caloso/genética , Proteínas de Transporte/genética , Ciclinas/deficiência , Ciclinas/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/patologia , Animais , Éxons , Estudos de Associação Genética , Homozigoto , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Insercional , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.
Assuntos
Sistemas CRISPR-Cas/genética , Marcação de Genes/métodos , Monofenol Mono-Oxigenase/genética , Mutação/genética , Alelos , Animais , Sequência de Bases , DNA/genética , Feminino , Vetores Genéticos/metabolismo , Genoma/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microinjeções , Dados de Sequência Molecular , Linhagem , Zigoto/metabolismoRESUMO
In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice harboring luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity through the use of other luciferases and/or by improving the biodistribution of their substrates in the animal body. Here we created two Cre-dependent reporter mice incorporating luciferases oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI; however, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the lights produced by oFluc and Akaluc were sufficiently spectrally distinct from each other for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.
RESUMO
Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.
Assuntos
Eletroporação , Zigoto , Humanos , Animais , Camundongos , Terapia com Eletroporação , Patrimônio Genético , Camundongos Knockout , MamíferosRESUMO
Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Técnicas de Introdução de Genes/métodos , Genes Reporter/genética , Modelos Animais , Modelos Genéticos , Animais , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Corpo Lúteo/metabolismo , Quinase 5 Dependente de Ciclina/fisiologia , Feminino , Expressão Gênica , Proteínas Luminescentes , Masculino , Camundongos Endogâmicos C57BL , Testículo/metabolismo , Proteína Vermelha FluorescenteRESUMO
Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. We have produced transgenic mice expressing both reverse tetracycline-controlled transactivator (rtTA) and transcriptional silencer (tTS) ubiquitously. Although the transgene products do not affect development of the mouse brain, one of the founder lines, TAS, showed ACC, suggesting transgenic disruption of endogenous gene(s). To identify the causative gene and its role in ACC, we performed pathological investigations of the brain and chromosomal mapping of foreign genes in TAS mice. Sixty-two percent of the heterozygous TAS mice showed ACC accompanied with formation of Probst bundles, as seen in human. Complete penetrance of ACC was observed in homozygous TAS mice. Furthermore, homozygous TAS fetuses revealed that ACC is a congenital anomaly. Moreover, axons of the corpus callosum were not repelled by the midline glial structures in TAS mice. These findings suggested that the causative gene for ACC is involved in critical steps in corpus callosum development. Multiple FISH analyses were performed to determine the site of transgene insertion. On 1-color FISH analyses, rtTA and tTS were detected on the A/B region of chromosome 18, suggesting cointegration of the transgenes. On 2-color FISH analyses, tTS signal was observed in a region from 9.3 to 16.9 Mb on chromosome 18. The TAS mice may serve as a useful model to identify a novel gene regulating corpus callosum development and to gain a new insight into molecular genetics of ACC.
Assuntos
Agenesia do Corpo Caloso , Cromossomos de Mamíferos/genética , Regulação da Expressão Gênica no Desenvolvimento , Transgenes , Animais , Axônios/metabolismo , Mapeamento Cromossômico , Loci Gênicos , Predisposição Genética para Doença , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neuroglia/metabolismo , Elementos Silenciadores Transcricionais , Tetraciclina/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).
Assuntos
Células-Tronco Embrionárias/fisiologia , Camundongos Endogâmicos C57BL , Animais , Técnicas de Cultura de Células , Linhagem Celular , Forma Celular , Células Cultivadas , Quimera/genética , Técnicas de Cocultura , Meios de Cultura/química , Células-Tronco Embrionárias/citologia , Marcação de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Recombinação GenéticaRESUMO
The in vivo imaging of mice makes it possible to analyze disease progress non-invasively through reporter gene expression. As the removal of hair improves the accuracy of in vivo imaging, gene-modified mice with a reporter gene are often crossed with Hos:HR-1 mutant mice homozygous for the spontaneous Hrhr mutation that exhibit a hair loss phenotype. However, it is time consuming to produce mice carrying both the reporter gene and mutant Hrhr gene by mating. In addition, there is a risk that genetic background of the gene-modified mice would be altered by mating. To resolve these issues, we established a simple method to generate hairless mice maintaining the original genetic background by CRISPR technology. First, we constructed the pX330 vector, which targets exon 3 of Hr. This DNA vector (5 ng/µl) was microinjected into the pronuclei of C57BL/6J mice. Induced Hr gene mutations were found in many founders (76.1%) and these mutations were heritable. Next, we performed in vivo imaging using these gene-modified hairless mice. As expected, luminescent objects in their body were detected by in vivo imaging. This study clearly showed that hairless mice could be simply generated by the CRISPR/Cas9 system, and this method may be useful for in vivo imaging studies with various gene-modified mice.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Diagnóstico por Imagem/métodos , Camundongos Pelados/genética , Terapia de Substituição Mitocondrial/métodos , Mutação , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , DNA/genética , Genes Reporter/genética , Vetores Genéticos , Camundongos Endogâmicos C57BL , Microinjeções , FenótipoRESUMO
We found a novel spontaneous mouse mutant with depigmentation in the ventral body, which we called White Spotting (WS) mouse. Genetic investigation revealed deletion of a > 1.2-Mb genomic region containing nine genes (Kit, Kdr, Srd5a3, Tmeme165, Clock, Pdcl2, Nmu, Exoc1, and Cep135). We designated this mutant allele Kit(WS). Interestingly, homozygous mutants (Kit(WS/WS)) showed a peri-implantation lethal phenotype. Expression analyses of these nine genes in blastocysts suggested that Exoc1 was a prime candidate for this phenotype. We produced Exoc1 knockout mice, and the same peri-implantation lethal phenotype was seen in Exoc1(-/-) embryos. In addition, the polygenic effect without Exoc1 was investigated in genome-edited Kit(WE) mice carrying the Mb-scale deletion induced by the CRISPR/Cas9 system. As Kit(WE/WE) embryos did not exhibit the abnormal phenotype, which was seen in Kit(WS/WS). We concluded that peri-implantation lethality in Kit(WS/WS) was caused by a monogenic defect of Exoc1.
Assuntos
Deleção Cromossômica , Deleção de Genes , Genes Letais , Fenótipo , Animais , Blastocisto/metabolismo , Mapeamento Cromossômico , Cruzamentos Genéticos , Masculino , Camundongos , Camundongos Knockout , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-kit/genética , Edição de RNA , Proteínas de Transporte VesicularRESUMO
Cre/loxP system-mediated site-specific recombination is utilized to study gene function in vivo. Successful conditional knockout of genes of interest is dependent on the availability of Cre-driver mice. We produced and characterized pancreatic ß cell-specific Cre-driver mice for use in diabetes mellitus research. The gene encoding Cre was inserted into the second exon of mouse Ins1 in a bacterial artificial chromosome (BAC). Five founder mice were produced by microinjection of linearized BAC Ins1-cre. The transgene was integrated between Mafa and the telomere on chromosome 15 in one of the founders, BAC Ins1-cre25. To investigate Cre-loxP recombination, BAC Ins1-cre25 males were crossed with two different Cre-reporters, R26R and R26GRR females. On gross observation, reporter signal after Cre-loxP recombination was detected exclusively in the adult pancreatic islets in both F1 mice. Immunohistological analysis indicated that Cre-loxP recombination-mediated reporter signal was colocalized with insulin in pancreatic islet cells of both F1 mice, but not with glucagon. Moreover, Cre-loxP recombination signal was already observed in the pancreatic islets at E13.5 in both F1 fetuses. Finally, we investigated ectopic Cre-loxP recombination for Ins1, because the ortholog Ins2 is also expressed in the brain, in addition to the pancreas. However, there was no Cre-loxP recombination-mediated reporter signal in the brain of both F1 mice. Our data suggest that BAC Ins1-cre25 mice are a useful Cre-driver C57BL/6N for pancreatic ß cell-specific Cre-loxP recombination, except for crossing with knock-in mice carrying floxed gene on chromosome 15.
Assuntos
Proteínas da Matriz Extracelular/genética , Células Secretoras de Insulina , Insulina/genética , Integrases/genética , Camundongos Transgênicos/genética , Proteína-Lisina 6-Oxidase/genética , Recombinação Genética/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Diabetes Mellitus/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The Cre/loxP system is a strategy for controlling temporal and/or spatial gene expression through genome alteration in mice. As successful Cre/loxP genome alteration depends on Cre-driver mice, Cre-reporter mice are essential for validation of Cre gene expression in vivo. In most Cre-reporter mouse strains, although the presence of reporter product indicates the expression of Cre recombinase, it has remained unclear whether a lack of reporter signal indicates either no Cre recombinase expression or insufficient reporter gene promoter activity. We produced a novel ROSA26 knock-in Cre-reporter C57BL/6N strain exhibiting green emission before and red after Cre-mediated recombination, designated as strain R26GRR. Ubiquitous green fluorescence and no red fluorescence were observed in R26GRR mice. To investigate the activation of tdsRed, EGFP-excised R26GRR, R26RR, mice were produced through the crossing of C57BL/6N mice with R26GRR/Ayu1-Cre F1 mice. R26RR mice showed extraordinarily strong red fluorescence in almost all tissues examined, suggesting ubiquitous activation of the second reporter in all tissues after Cre/loxP recombination. Moreover, endothelial cell lineage and pancreatic islet-specific expression of red fluorescence were detected in R26GRR/Tie2-Cre F1 mice and R26GRR /Ins1-Cre F1 mice, respectively. These results indicated that R26GRR mice are a useful novel Cre-reporter mouse strain. In addition, R26GRR mice with a pure C57BL/6N background represent a valuable source of green-to-red photoconvertible cells following Cre/loxP recombination for application in transplantation studies. The R26GRR mouse strain will be available from RIKEN BioResource Center (http://www.brc.riken.jp/lab/animal/en/).
Assuntos
Expressão Gênica , Genes Reporter/genética , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Integrases/genética , Integrases/metabolismo , Animais , Células Cultivadas , Células-Tronco Embrionárias , Células Endoteliais/metabolismo , Feminino , Técnicas de Introdução de Genes , Ilhotas Pancreáticas/metabolismo , Proteínas Luminescentes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Recombinação Genética , Ubiquitinação , Proteína Vermelha FluorescenteRESUMO
We found 6 spontaneous mutant mice with long pelage hair in our ICR breeding colony. The abnormal trait was restricted to long hair in these mice, which we named moja. They were fertile and showed the same growth and behavior as wild-type mice. To investigate the manner of the genetic inheritance of the moja allele, offspring were bred by mating the moja mice; all offspring had long pelage hair. Furthermore, we performed a reciprocal cross between moja mice and wild-type ICR mice with normal hair. All offspring exhibited normal hair suggesting an autosomal recessive inheritance of the trait. The moja/moja hair phenotype was maintained in skin grafted onto nude mice, suggesting that circulating or diffusible humoral factors regulating the hair cycle are not involved in the abnormal trait. The phenotype of moja/moja mice is similar to that of Fgf5-deficient mice. Therefore, we examined the expression of Fgf5 by RT-PCR in moja/moja mice. As expected, no Fgf5 expression was found in moja/moja mouse skin. PCR and DNA sequence analyses were performed to investigate the structure of the Fgf5 gene. We found a deletion of a 9.3-kb region in the Fgf5 gene including exon 3 and its 5' and 3' flanking sequences. Interestingly, the genomic deletion site showed insertion of a 498-bp early transposon element long terminal repeat. Taken together, these results suggest that the long hair mutation of moja/moja mice is caused by disruption of Fgf5 mediated by insertion of a retrotransposon.
Assuntos
Fator 5 de Crescimento de Fibroblastos/genética , Cabelo/crescimento & desenvolvimento , Camundongos/genética , Retroelementos , Animais , Sequência de Bases , Cruzamentos Genéticos , Feminino , Deleção de Genes , Cabelo/fisiologia , Masculino , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Camundongos Nus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
As the phenotype of a given single-gene mutation in mice is modulated by the genetic background of the inbred strain, embryonic stem (ES) cells derived from various inbred mouse strains are required to produce gene-targeted mice without the need for backcrossing and for detailed analysis of gene function in vivo. Here, we performed a comparative investigation of the effects of three culture conditions, LIF + KSR/ES medium described previously, High LIF + KSR/ES medium and iSTEM + LIF medium containing three inhibitors of glycogen synthase kinase 3, mitogen-activated protein kinase kinase, and fibroblast growth factor receptor signaling (3i), on the establishment of germline-competent ES cells derived from strains BALB/c and NZB mice. The results indicated that LIF + KSR/ES medium was permissive for the derivation of ES cells from NZB mice, which contribute to the somatic lineage in vivo, but not to the germline lineage. In contrast, ES cells that contribute to the makeup of chimeric mice were not propagated from blastocysts of BALB/c mice. Both germline and somatic competency were improved by increased LIF concentration in cultures of BALB/c ES cells, although we failed to establish germline-competent NZB ES cells using the same concentration of LIF. Unexpectedly, iSTEM + LIF medium containing 3i showed a negative effect on the derivation of NZB ES cells with normal chromosome numbers, but not on the maintenance of previously established ES cells. Our findings suggest that the stability of pluripotency in the inner cell mass isolated from blastocyst embryos may differ according to the genetic background of inbred mouse strains, and that although the concentration of LIF is a determinant for authentic pluripotency, including germline and somatic competency in BALB/c ES cells, additional factor(s) are required for commitment to germline lineage independent of somatic lineage in NZB ES cells.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Embrionárias/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB , Animais , Células Cultivadas , Quimera , Técnicas de Cocultura , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In order to save natural resources and supply good fishes, it is important to improve fish-farming techniques. The survival rate of fish fry appears to become higher when powders of foraminifer limestone are submerged at the bottom of fish-farming fields, where bacterial biofilms often grow. The observations suggest that forming biofilms can benefit to keep health status of breeding fishes. We employed culture-based methods for the identification and characterization of biofilm-forming bacteria and assessed the application of their properties for fish farming. Fifteen bacterial strains were isolated from the biofilm samples collected from fish farm sediments. The 16S rRNA gene sequences indicated that these bacteria belonged to the genera, Pseudoalteromonas (seven strains), Vibrio (seven strains) and Halomonas (one strain). It was found that Pseudoalteromonas strains generally formed robust biofilms in a laboratory condition and produced extracellular proteases in a biofilm-dependent manner. The results suggest that Pseudoalteromonas bacteria, living in the biofilm community, contribute in part to remove excess proteineous matters from the sediment sludge of fish farms.