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1.
Proc Natl Acad Sci U S A ; 116(3): 960-969, 2019 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-30593561

RESUMEN

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.


Asunto(s)
Envejecimiento , ARN Helicasas DEAD-box , Homocigoto , Mutación Missense , Ribonucleasa III , Envejecimiento/genética , Envejecimiento/metabolismo , Sustitución de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Masculino , Ratones , Fosforilación/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo
2.
Gastroenterology ; 159(6): 2146-2162.e33, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32805281

RESUMEN

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.


Asunto(s)
Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Neoplasias Experimentales/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Aurora Quinasa B/metabolismo , Azoximetano/toxicidad , Carcinogénesis/genética , Línea Celular Tumoral , Colon/citología , Colon/patología , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/patología , Análisis Citogenético , Dextranos/toxicidad , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/patología , Organoides , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética
3.
Genome Res ; 28(4): 432-447, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29567676

RESUMEN

The cancer-risk-associated rs6983267 single nucleotide polymorphism (SNP) and the accompanying long noncoding RNA CCAT2 in the highly amplified 8q24.21 region have been implicated in cancer predisposition, although causality has not been established. Here, using allele-specific CCAT2 transgenic mice, we demonstrate that CCAT2 overexpression leads to spontaneous myeloid malignancies. We further identified that CCAT2 is overexpressed in bone marrow and peripheral blood of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) patients. CCAT2 induces global deregulation of gene expression by down-regulating EZH2 in vitro and in vivo in an allele-specific manner. We also identified a novel non-APOBEC, non-ADAR, RNA editing at the SNP locus in MDS/MPN patients and CCAT2-transgenic mice. The RNA transcribed from the SNP locus in malignant hematopoietic cells have different allelic composition from the corresponding genomic DNA, a phenomenon rarely observed in normal cells. Our findings provide fundamental insights into the functional role of rs6983267 SNP and CCAT2 in myeloid malignancies.


Asunto(s)
Proliferación Celular/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , ARN Largo no Codificante/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Enfermedades Mielodisplásicas-Mieloproliferativas/patología , Polimorfismo de Nucleótido Simple/genética , Edición de ARN/genética
4.
Nature ; 517(7536): 626-30, 2015 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-25409149

RESUMEN

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.


Asunto(s)
Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Linfoma/metabolismo , Linfoma/patología , Proteína p53 Supresora de Tumor/deficiencia , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genes Supresores de Tumor , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/uso terapéutico , Linfoma/tratamiento farmacológico , Linfoma/genética , Masculino , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína 3 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Calcitonina/metabolismo , Timo/metabolismo , Timo/patología , Transactivadores/genética , Transactivadores/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(5): E572-81, 2014 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-24449888

RESUMEN

The roles of microRNAs (miRNAs) and the miRNA processing machinery in the regulation of stem cell biology are not well understood. Here, we show that the p53 family member and p63 isoform, ΔNp63, is a transcriptional activator of a cofactor critical for miRNA processing (DGCR8). This regulation gives rise to a unique miRNA signature resulting in reprogramming cells to multipotency. Strikingly, ΔNp63(-/-) epidermal cells display profound defects in terminal differentiation and express a subset of markers and miRNAs present in embryonic stem cells and fibroblasts induced to pluripotency using Yamanaka factors. Moreover, ΔNp63(-/-) epidermal cells transduced with an inducible DGCR8 plasmid can differentiate into multiple cell fates in vitro and in vivo. We found that human primary keratinocytes depleted of ΔNp63 or DGCR8 can be reprogrammed in 6 d and express a unique miRNA and gene expression signature that is similar but not identical to human induced pluripotent stem cells. Our data reveal a role for ΔNp63 in the transcriptional regulation of DGCR8 to reprogram adult somatic cells into multipotent stem cells.


Asunto(s)
Regulación hacia Abajo/genética , Queratinocitos/metabolismo , Células Madre Multipotentes/citología , Fosfoproteínas/genética , Proteínas/genética , Proteínas de Unión al ARN/genética , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Adulto , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Quimera , Embrión de Mamíferos/citología , Células Epidérmicas , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Queratinocitos/citología , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Multipotentes/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosfoproteínas/deficiencia , Fosfoproteínas/metabolismo , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transactivadores/deficiencia , Transactivadores/metabolismo , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo
7.
Dev Biol ; 393(1): 3-9, 2014 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-24984260

RESUMEN

Tyrosinase is the rate-limiting enzyme for the production of melanin pigmentation. In the mouse and other animals, homozygous null mutations in the Tyrosinase gene (Tyr) result in the absence of pigmentation, i.e. albinism. Here we used the CRISPR/Cas9 system to generate mono- and bi-allelic null mutations in the Tyr locus by zygote injection of two single-guide and Cas9 RNAs. Injection into C57BL/6N wild-type embryos resulted in one completely albino founder carrying two different Tyr mutations. In addition, three pigmentation mosaics and fully pigmented littermates were obtained that transmitted new mutant Tyr alleles to progeny in test crosses with albinos. Injection into Tyr heterozygous (B6CBAF1/J×FVB/NJ) zygotes resulted in the generation of numerous albinos and also mice with a graded range of albino mosaicism. Deep sequencing revealed that the majority of the albinos and the mosaics had more than two new mutant alleles. These visual phenotypes and molecular genotypes highlight the somatic mosaicism and allele complexity in founders that occurs for targeted genes during CRISPR/Cas9-mediated mutagenesis by zygote injection in mice.


Asunto(s)
Albinismo/genética , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica/métodos , Monofenol Monooxigenasa/genética , Mosaicismo/embriología , Pigmentación/genética , Alelos , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Melaninas/genética , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Mutación , ARN Mensajero/genética , Análisis de Secuencia de ADN , Cigoto/citología , ARN Pequeño no Traducido
8.
Nature ; 453(7192): 223-7, 2008 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-18362916

RESUMEN

The neuronal repressor REST (RE1-silencing transcription factor; also called NRSF) is expressed at high levels in mouse embryonic stem (ES) cells, but its role in these cells is unclear. Here we show that REST maintains self-renewal and pluripotency in mouse ES cells through suppression of the microRNA miR-21. We found that, as with known self-renewal markers, the level of REST expression is much higher in self-renewing mouse ES cells than in differentiating mouse ES (embryoid body, EB) cells. Heterozygous deletion of Rest (Rest+/-) and its short-interfering-RNA-mediated knockdown in mouse ES cells cause a loss of self-renewal-even when these cells are grown under self-renewal conditions-and lead to the expression of markers specific for multiple lineages. Conversely, exogenously added REST maintains self-renewal in mouse EB cells. Furthermore, Rest+/- mouse ES cells cultured under self-renewal conditions express substantially reduced levels of several self-renewal regulators, including Oct4 (also called Pou5f1), Nanog, Sox2 and c-Myc, and exogenously added REST in mouse EB cells maintains the self-renewal phenotypes and expression of these self-renewal regulators. We also show that in mouse ES cells, REST is bound to the gene chromatin of a set of miRNAs that potentially target self-renewal genes. Whereas mouse ES cells and mouse EB cells containing exogenously added REST express lower levels of these miRNAs, EB cells, Rest+/- ES cells and ES cells treated with short interfering RNA targeting Rest express higher levels of these miRNAs. At least one of these REST-regulated miRNAs, miR-21, specifically suppresses the self-renewal of mouse ES cells, corresponding to the decreased expression of Oct4, Nanog, Sox2 and c-Myc. Thus, REST is a newly discovered element of the interconnected regulatory network that maintains the self-renewal and pluripotency of mouse ES cells.


Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Biomarcadores , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Represoras/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
9.
Exp Dermatol ; 21(3): 217-20, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22379968

RESUMEN

Strains of mice vary in their susceptibility to ultra-violet (UV) radiation-induced skin tumors. Some strains of hairless mice (homozygous for the spontaneous Hr(hr) mutation) are particularly susceptible to these tumors. The skin tumors that develop in hairless mice resemble, both at the morphologic and molecular levels, UV-induced squamous cell carcinomas (SCC) and their precursors in human. The most commonly employed hairless mice belong to the SKH1 stock. However, these mice are outbred and their genetic background is not characterized, which makes them a poor model for genetic studies. We have developed a new inbred strain from outbred SKH1 mice that we named SKHIN/Sprd (now at generation F31). In order to characterize the genetic background of this new strain, we genotyped a cohort of mice at F30 with 92 microsatellites and 140 single nucleotide polymorphisms (SNP) evenly distributed throughout the mouse genome. We also exposed SKHIN/Sprd mice to chronic UV irradiation and showed that they are as susceptible to UV-induced skin carcinogenesis as outbred SKH1 mice. In addition, we proved that, albeit with low efficiency, inbred SKHIN/Sprd mice are suitable for transgenic production by classical pronuclear microinjection. This new inbred strain will be useful for the development of transgenic and congenic strains on a hairless inbred background as well as the establishment of syngeneic tumor cell lines. These new tools can potentially help elucidate a number of features of the cutaneous response to UV irradiation in humans, including the effect of genetic background and modifier genes.


Asunto(s)
Ratones Pelados/genética , Modelos Animales , Neoplasias Inducidas por Radiación/genética , Neoplasias Cutáneas/genética , Animales , Modelos Animales de Enfermedad , Ratones , Neoplasias Inducidas por Radiación/etiología , Rayos Ultravioleta
11.
Nat Commun ; 12(1): 5389, 2021 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-34508101

RESUMEN

Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.


Asunto(s)
Carcinosarcoma/genética , Proteínas Portadoras/genética , Neoplasias Mamarias Experimentales/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Mama/patología , Carcinosarcoma/patología , Proteínas Portadoras/metabolismo , Ensayos Clínicos como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-met/genética , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Secuenciación Completa del Genoma , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Methods Mol Biol ; 2066: 163-169, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31512216

RESUMEN

Genetically modified mice can be generated using a variety of methods. Depending on the method used, the breeding strategy must be modified not only for the initial founding generation of mouse, but to establish individual mouse lines as well. Transgenic founder mice are each unique and must be outcrossed to initially establish the line. Mice that have been targeted using embryonic stem cells will need to be tested for germ line transmission of the modified gene prior to establishing a line. Mice that have been targeted using CRISPR/Cas9 gene-editing endonucleases will often be mosaic, and thus, also require testing for germ line transmission of the mutation of interest.


Asunto(s)
Cruzamiento/métodos , Sistemas CRISPR-Cas/genética , Células Madre Embrionarias , Ratones Transgénicos/genética , Animales , Células Germinativas/crecimiento & desarrollo , Ratones , Mutación/genética , ARN Guía de Kinetoplastida/genética , Cigoto/crecimiento & desarrollo
13.
Methods Mol Biol ; 2066: 133-148, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31512214

RESUMEN

Prior to generating a new mouse model, it is important to plan the method that will be used to detect which of the mice generated have the mutation(s) desired. Nearly, all types of mutations may be detected using PCR. However, the choice of primers will differ depending upon the method used to generate the model. Transgenic mice should be genotyped across a unique junction fragment. Targeted ES cells used to generate knock-out or knock-in mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Targeting in ES cells can also be detected using a genomic Southern blot. Mice targeted using CRISPR/Cas9 should have the region of interest amplified using PCR, and then be assessed for size changes (for large changes in sequence) by Surveyor Assay (for gene knock-out and point mutations) and/or sequenced to verify the mutation. Each of these models has a unique requirement for genotyping, and failure to understand the requirements can easily lead to loss of the gene in subsequent generations.


Asunto(s)
Animales Modificados Genéticamente/genética , Sistemas CRISPR-Cas/genética , Técnicas de Inactivación de Genes/métodos , Ratones Transgénicos , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Células Madre Embrionarias/metabolismo , Genotipo , Ratones , Mutación/genética
14.
Genes (Basel) ; 11(6)2020 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-32521708

RESUMEN

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.


Asunto(s)
Animales Modificados Genéticamente/genética , Sistemas CRISPR-Cas/genética , Ingeniería Genética , ARN Guía de Kinetoplastida/genética , Animales , Blastocisto/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutación/genética
15.
Lab Anim (NY) ; 38(6): 195-201, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19455165

RESUMEN

Computer-based training is potentially a useful means of gaining proficiency in various aspects of laboratory animal science. The authors present an overview of the AALAS Learning Library (ALL), an internet-based training system that was established in 2003 and is widely used for technician certification training and for IACUC-mandated training. To evaluate the effectiveness of the ALL as a tool for general training and for achieving certification, the AALAS Online Learning Committee initiated a review of the online courses. The authors analyzed the numbers of users who accessed different types of courses and completed exams in those courses. They also correlated ALL usage with pass rates in technician certification exams. Results suggest that the ALL is a highly effective method of training, particularly for technician certification.


Asunto(s)
Técnicos de Animales/educación , Bienestar del Animal/normas , Simulación por Computador , Instrucción por Computador/métodos , Ciencia de los Animales de Laboratorio/educación , Evaluación de Programas y Proyectos de Salud , Acreditación , Animales , Animales de Laboratorio , Humanos , Ciencia de los Animales de Laboratorio/normas , Competencia Profesional , Sociedades Científicas
16.
Cell Rep ; 27(3): 900-915.e5, 2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30995485

RESUMEN

In the mouse retina, more than 30 retinal ganglion cell (RGC) subtypes have been classified based on a combined metric of morphological and functional characteristics. RGCs arise from a common pool of retinal progenitor cells during embryonic stages and differentiate into mature subtypes in adult retinas. However, the cellular and molecular mechanisms controlling formation and maturation of such remarkable cellular diversity remain unknown. Here, we demonstrate that T-box transcription factor T-brain 1 (Tbr1) is expressed in two groups of morphologically and functionally distinct RGCs: the orientation-selective J-RGCs and a group of OFF-sustained RGCs with symmetrical dendritic arbors. When Tbr1 is genetically ablated during retinal development, these two RGC groups cannot develop. Ectopically expressing Tbr1 in M4 ipRGCs during development alters dendritic branching and density but not the inner plexiform layer stratification level. Our data indicate that Tbr1 plays critical roles in regulating the formation and dendritic morphogenesis of specific RGC types.


Asunto(s)
Células Ganglionares de la Retina/metabolismo , Proteínas de Dominio T Box/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Axones/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Toxina del Cólera/toxicidad , Dendritas/fisiología , Embrión de Mamíferos/metabolismo , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Potasio/farmacología , Retina/crecimiento & desarrollo , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Proteínas de Dominio T Box/genética
17.
JCI Insight ; 4(6)2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-30721150

RESUMEN

Airway mucin secretion is necessary for ciliary clearance of inhaled particles and pathogens but can be detrimental in pathologies such as asthma and cystic fibrosis. Exocytosis in mammals requires a Munc18 scaffolding protein, and airway secretory cells express all 3 Munc18 isoforms. Using conditional airway epithelial cell-deletant mice, we found that Munc18a has the major role in baseline mucin secretion, Munc18b has the major role in stimulated mucin secretion, and Munc18c does not function in mucin secretion. In an allergic asthma model, Munc18b deletion reduced airway mucus occlusion and airflow resistance. In a cystic fibrosis model, Munc18b deletion reduced airway mucus occlusion and emphysema. Munc18b deficiency in the airway epithelium did not result in any abnormalities of lung structure, particle clearance, inflammation, or bacterial infection. Our results show that regulated secretion in a polarized epithelial cell may involve more than one exocytic machine at the apical plasma membrane and that the protective roles of mucin secretion can be preserved while therapeutically targeting its pathologic roles.


Asunto(s)
Asma/metabolismo , Mucinas/metabolismo , Proteínas Munc18/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Exocitosis , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Munc18/genética , Mucosa Respiratoria/patología , Transcriptoma
18.
Autophagy ; 14(4): 637-653, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29368982

RESUMEN

Among the 3 GTPases in the DIRAS family, DIRAS3/ARHI is the best characterized. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. Diras3 has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40-50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.


Asunto(s)
Autofagia/fisiología , Carcinoma Epitelial de Ovario/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neoplasias Ováricas/metabolismo , Animales , Línea Celular Tumoral , Femenino , GTP Fosfohidrolasas/metabolismo , Neoplasias Ováricas/patología , Ovario/metabolismo , Proteínas Supresoras de Tumor/metabolismo
19.
Front Cell Neurosci ; 12: 365, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30386211

RESUMEN

Multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS) is the leading cause of non-traumatic neurological disability in young adults. Immune mediated destruction of myelin and oligodendrocytes is considered the primary pathology of MS, but progressive axonal loss is the major cause of neurological disability. In an effort to understand microglia function during CNS inflammation, our laboratory focuses on the fractalkine/CX3CR1 signaling as a regulator of microglia neurotoxicity in various models of neurodegeneration. Fractalkine (FKN) is a transmembrane chemokine expressed in the CNS by neurons and signals through its unique receptor CX3CR1 present in microglia. During experimental autoimmune encephalomyelitis (EAE), CX3CR1 deficiency confers exacerbated disease defined by severe inflammation and neuronal loss. The CX3CR1 human polymorphism I249/M280 present in ∼20% of the population exhibits reduced adhesion for FKN conferring defective signaling whose role in microglia function and influence on neurons during MS remains unsolved. The aim of this study is to assess the effect of weaker signaling through hCX3CR1I249/M280 during EAE. We hypothesize that dysregulated microglial responses due to impaired CX3CR1 signaling enhance neuronal/axonal damage. We generated an animal model replacing the mouse CX3CR1 locus for the hCX3CR1I249/M280 variant. Upon EAE induction, these mice exhibited exacerbated EAE correlating with severe inflammation and neuronal loss. We also observed that mice with aberrant CX3CR1 signaling are unable to produce FKN and ciliary neurotrophic factor during EAE in contrast to wild type mice. Our results provide validation of defective function of the hCX3CR1I249/M280 variant and the foundation to broaden the understanding of microglia dysfunction during neuroinflammation.

20.
Cell Biosci ; 7: 58, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29118970

RESUMEN

BACKGROUND: The 14-3-3 family of proteins have been reported to play an important role in development in various mouse models, but the context specific developmental functions of 14-3-3ζ remain to be determined. In this study, we identified a context specific developmental function of 14-3-3ζ. RESULTS: Targeted deletion of 14-3-3ζ in the C57Bl/6J murine genetic background led to neonatal lethality due to respiratory distress and could be rescued by out-breeding to the CD-1 or backcrossing to the FVB/NJ congenic background. Histological analysis of lung sections from 18.5 days post coitum embryos (dpc) showed that 14-3-3ζ-/- lung development is arrested at the pseudoglandular stage and exhibits vascular defects. The expression of miR-126, an endothelial-specific miRNA known to regulate lung vascular integrity was down-regulated in the lungs of the 14-3-3ζ-/- embryos in the C57Bl/6J background as compared to their wild-type counterparts. Loss of 14-3-3ζ in endothelial cells inhibited the angiogenic capability of the endothelial cells as determined by both trans-well migration assays and tube formation assays and these defects could be rescued by re-expressing miR-126. Mechanistically, loss of 14-3-3ζ led to reduced Erk1/2 phosphorylation resulting in attenuated binding of the transcription factor Ets2 on the miR-126 promoter which ultimately reduced expression of miR-126. CONCLUSION: Our data demonstrates that miR-126 is an important angiogenesis regulator that functions downstream of 14-3-3ζ and downregulation of miR-126 plays a critical role in 14-3-3ζ-loss induced defects in lung vasculature in the C57Bl/6J genetic background.

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